Modulators and methods of use

ABSTRACT

Novel modulators, including antibodies and derivatives thereof, and methods of such modulators to treat hyperproliferative disorders are provided.

CROSS REFERENCED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 13/992,599,filed Jun. 7, 2013, now U.S. Pat. No. 9,320,812, issued Apr. 26, 2016,which is a national stage application of PCT/US2011/063831 filed Dec. 7,2011, which is a continuation-in-part of PCT/US2011/050451 and claimspriority to U.S. Provisional Application No. 61/421,157 filed Dec. 8,2010, each of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 22, 2011, isnamed 11200PCT.txt and is 80,102 bytes in size.

FIELD OF THE INVENTION

This application generally relates to novel compositions and methods oftheir use in preventing, treating or ameliorating hyperproliferativedisorders and any expansion, recurrence, relapse or metastasis thereof.In a broad aspect, the present invention relates to the use of ephrin-Aligand (EFNA) modulators, including anti-EFNA antibodies and fusionconstructs, for the treatment or prophylaxis of neoplastic disorders.Particularly preferred embodiments of the present invention provide forthe use of such EFNA modulators for the immunotherapeutic treatment ofmalignancies comprising a reduction in tumor initiating cell frequency.

BACKGROUND OF THE INVENTION

Stem and progenitor cell differentiation and cell proliferation arenormal ongoing processes that act in concert to support tissue growthduring organogenesis and cell replacement and repair of most tissuesduring the lifetime of all living organisms. Differentiation andproliferation decisions are often controlled by numerous factors andsignals that are balanced to maintain cell fate decisions and tissuearchitecture. Normal tissue architecture is largely maintained by cellsresponding to microenvironmental cues that regulate cell division andtissue maturation. Accordingly, cell proliferation and differentiationnormally occurs only as necessary for the replacement of damaged ordying cells or for growth. Unfortunately, disruption of cellproliferation and/or differentiation can result from a myriad of factorsincluding, for example, the under- or overabundance of various signalingchemicals, the presence of altered microenvironments, genetic mutationsor some combination thereof. When normal cellular proliferation and/ordifferentiation is disturbed or somehow disrupted it can lead to variousdiseases or disorders including hyperproliferative disorders such ascancer.

Conventional treatments for cancer include chemotherapy, radiotherapy,surgery, immunotherapy (e.g., biological response modifiers, vaccines ortargeted therapeutics) or combinations thereof. Sadly, far too manycancers are non-responsive or minimally responsive to such conventionaltreatments leaving few options for patients. For example, in somepatients certain cancers exhibit gene mutations that render themnon-responsive despite the general effectiveness of selected therapies.Moreover, depending on the type of cancer some available treatments,such as surgery, may not be viable alternatives. Limitations inherent incurrent standard of care therapeutics are particularly evident whenattempting to care for patients who have undergone previous treatmentsand have subsequently relapsed. In such cases the failed therapeuticregimens and resulting patient deterioration may contribute torefractory tumors which often manifest themselves as a more aggressivedisease that ultimately proves to be incurable. Although there have beengreat improvements in the diagnosis and treatment of cancer over theyears, overall survival rates for many solid tumors have remainedlargely unchanged due to the failure of existing therapies to preventrelapse, tumor recurrence and metastases. Thus, it remains a challengeto develop more targeted and potent therapies.

One promising area of research involves the use of targeted therapeuticsto go after the tumorigenic “seed” cells that appear to underlie manycancers. To that end most solid tissues are now known to contain adult,tissue-resident stem cell populations generating the differentiated celltypes that comprise the majority of that tissue. Tumors arising in thesetissues similarly consist of heterogeneous populations of cells thatalso arise from stem cells, but differ markedly in their overallproliferation and organization. While it is increasingly recognized thatthe majority of tumor cells have a limited ability to proliferate, aminority population of cancer cells (commonly known as cancer stem cellsor CSC) have the exclusive ability to extensively self-renew therebyenabling an inherent tumor reinitiating capacity. More specifically, thecancer stem cell hypothesis proposes that there is a distinct subset ofcells (i.e. CSC) within each tumor (approximately 0.1-10%) that iscapable of indefinite self-renewal and of generating tumor cellsprogressively limited in their replication capacity as a result ofdifferentiation to tumor progenitor cells and, subsequently, toterminally differentiated tumor cells.

In recent years it has become more evident these CSC (also known astumor perpetuating cells or TPC) might be more resistant to traditionalchemotherapeutic agents or radiation and thus persist after standard ofcare clinical therapies to later fuel the growth of refractory tumors,secondary tumors and promote metastases. Moreover, growing evidencesuggests that pathways that regulate organogenesis and/or theself-renewal of normal tissue-resident stem cells are deregulated oraltered in CSC, resulting in the continuous expansion of self-renewingcancer cells and tumor formation. See generally Al-Hajj et al., 2004,PMID: 15378087; and Dalerba et al., 2007, PMID: 17548814; each of whichis incorporated herein in its entirety by reference. Thus, theeffectiveness of traditional, as well as more recent targeted treatmentmethods, has apparently been limited by the existence and/or emergenceof resistant cancer cells that are capable of perpetuating the cancereven in face of these diverse treatment methods. Huff et al., EuropeanJournal of Cancer 42: 1293-1297 (2006) and Zhou et al., Nature ReviewsDrug Discovery 8: 806-823 (2009) each of which is incorporated herein inits entirety by reference. Such observations are confirmed by theconsistent inability of traditional debulking agents to substantiallyincrease patient survival when suffering from solid tumors, and throughthe development of an increasingly sophisticated understanding as to howtumors grow, recur and metastasize. Accordingly, recent strategies fortreating neoplastic disorders have recognized the importance ofeliminating, depleting, silencing or promoting the differentiation oftumor perpetuating cells so as to diminish the possibility of tumorrecurrence, metastasis or patient relapse.

Efforts to develop such strategies have incorporated recent workinvolving non-traditional xenograft (NTX) models, wherein primary humansolid tumor specimens are implanted and passaged exclusively inimmunocompromised mice. In numerous cancers such techniques confirm theexistence of a subpopulation of cells with the unique ability togenerate heterogeneous tumors and fuel their growth indefinitely. Aspreviously hypothesized, work in NTX models has confirmed thatidentified CSC subpopulations of tumor cells appear more resistant todebulking regimens such as chemotherapy and radiation, potentiallyexplaining the disparity between clinical response rates and overallsurvival. Further, employment of NTX models in CSC research has sparkeda fundamental change in drug discovery and preclinical evaluation ofdrug candidates that may lead to CSC-targeted therapies having a majorimpact on tumor recurrence and metastasis thereby improving patientsurvival rates. While progress has been made, inherent technicaldifficulties associated with handling primary and/or xenograft tumortissue, along with a lack of experimental platforms to characterize CSCidentity and differentiation potential, pose major challenges. As such,there remains a substantial need to selectively target cancer stem cellsand develop diagnostic, prophylactic or therapeutic compounds or methodsthat may be used in the treatment, prevention and/or management ofhyperproliferative disorders.

SUMMARY OF THE INVENTION

These and other objectives are provided for by the present inventionwhich, in a broad sense, is directed to methods, compounds, compositionsand articles of manufacture that may be used in the treatment of EFNAassociated disorders (e.g., hyperproliferative disorders or neoplasticdisorders). To that end, the present invention provides novel EFNA (orephrin-A ligand) modulators that effectively target tumor cells orcancer stem cells and may be used to treat patients suffering from awide variety of malignancies. As will be discussed in more detailherein, there are presently six known ephrin-A ligands (i.e., EFNAs 1-6)and the disclosed modulators may comprise or associate with any one, ormore than one, ephrin-A ligand. Moreover, in certain embodiments thedisclosed EFNA modulators may comprise any compound that recognizes,competes, agonizes, antagonizes, interacts, binds or associates with anEFNA polypeptide, its receptor or its gene and modulates, adjusts,alters, changes or modifies the impact of the EFNA protein on one ormore physiological pathways. Thus, in a broad sense the presentinvention is directed to isolated EFNA modulators. In preferredembodiments the invention is more particularly directed to isolatedEFNA1 modulators or isolated EFNA4 modulators (i.e., modulators thatcomprise or associate with at least EFNA1 or EFNA4). Moreover, asdiscussed extensively below such modulators may be used to providepharmaceutical compositions.

In selected embodiments of the invention, EFNA modulators may comprisean ephrin-A ligand itself or fragments thereof, either in an isolatedform or fused or associated with other moieties (e.g., Fc-EFNA, PEG-EFNAor EFNA associated with a targeting moiety). In other selectedembodiments EFNA modulators may comprise EFNA antagonists which, for thepurposes of the instant application, shall be held to mean any constructor compound that recognizes, competes, interacts, binds or associateswith EFNA and neutralizes, eliminates, reduces, sensitizes, reprograms,inhibits or controls the growth of neoplastic cells including tumorinitiating cells. In preferred embodiments the EFNA modulators of theinstant invention comprise anti-EFNA antibodies, or fragments orderivatives thereof, that have unexpectedly been found to silence,neutralize, reduce, decrease, deplete, moderate, diminish, reprogram,eliminate, or otherwise inhibit the ability of tumor initiating cells topropagate, maintain, expand, proliferate or otherwise facilitate thesurvival, recurrence, regeneration and/or metastasis of neoplasticcells. In particularly preferred embodiments the antibodies orimmunoreactive fragments may be associated with or conjugated to one ormore anti-cancer agents.

In one embodiment the EFNA modulator may comprise a humanized antibodywherein said antibody comprises a heavy chain variable region amino acidsequence selected from the group consisting of SEQ ID NO: 149, SEQ IDNO: 153, SEQ ID NO: 157 and SEQ ID NO: 161 and a light chain variableregion amino acid sequence selected from the group consisting of SEQ IDNO: 151, SEQ ID NO: 155, SEQ ID NO: 159 and SEQ ID NO: 163. In otherpreferred embodiments the invention will be in the form of a compositioncomprising a humanized antibody selected from the group consisting ofhSC4.5, hSC4.15, hSC4.22 and hSC4.47 and a pharmaceutically acceptablecarrier. In another preferred embodiment the EFNA modulator may comprisean antibody that comprises one or more CDRs from FIG. 7A (SEQ ID NOS:8-59 and 70-95). Preferably the antibody comprising at least one CDRfrom FIG. 7A will comprise a humanized antibody.

In certain other embodiments the invention will comprise an EFNAmodulator that reduces the frequency of tumor initiating cells uponadministration to a subject. Preferably the reduction in frequency willbe determined using in vitro or in vivo limiting dilution analysis. Inparticularly preferred embodiments such analysis may be conducted usingin vivo limiting dilution analysis comprising transplant of live humantumor cells into immunocompromised mice. Alternatively, the limitingdilution analysis may be conducted using in vitro limiting dilutionanalysis comprising limiting dilution deposition of live human tumorcells into in vitro colony supporting conditions. In either case, theanalysis, calculation or quantification of the reduction in frequencywill preferably comprise the use of Poisson distribution statistics toprovide an accurate accounting. It will be appreciated that, while suchquantification methods are preferred, other, less labor intensivemethodology such as flow cytometry or immunohistochemistry may also beused to provide the desired values and, accordingly, are expresslycontemplated as being within the scope of the instant invention. In suchcases the reduction in frequency may be determined using flow cytometricanalysis or immunohistochemical detection of tumor cell surface markersknown to enrich for tumor initiating cells.

As such, in another preferred embodiment of the instant inventioncomprises a method of treating an EFNA associated disorder comprisingadministering a therapeutically effective amount of an EFNA modulator toa subject in need thereof whereby the frequency of tumor initiatingcells is reduced. Again, the reduction in the tumor initiating cellfrequency will preferably be determined using in vitro or in vivolimiting dilution analysis.

In this regard it will be appreciated that the present invention isbased, at least in part, upon the discovery that EFNA polypeptides (andparticularly EFNA4 as discussed below) are associated with tumorperpetuating cells (i.e., cancer stem cells) that are involved in theetiology of various neoplasia. More specifically, the instantapplication unexpectedly demonstrates that the administration of variousexemplary EFNA modulators can mediate, reduce, inhibit or eliminatetumorigenic signaling by tumor initiating cells (i.e., reduce thefrequency of tumor initiating cells). This reduced signaling, whether byreduction, elimination, reprogramming or silencing of the tumorinitiating cells or by modifying tumor cell morphology (e.g., induceddifferentiation, niche disruption), in turn allows for the moreeffective treatment of EFNA associated disorders by inhibitingtumorigenesis, tumor maintenance, expansion and/or metastasis andrecurrence. In other embodiments the disclosed modulators may promote,support or otherwise enhance EFNA mediated signaling that may limit orrestrain tumor growth. In other embodiments the disclosed modulators mayinterfere, suppress or otherwise retard EFNA mediated signaling that mayfuel tumor growth. Further, as will be discussed in more detail below,EFNA polypeptides are involved in generating adhesive and repulsiveforces between cells through integrin and cytoskeleton rearrangements.Intervention in such intercellular interactions, using the novel EFNAmodulators described herein, may thereby ameliorate a disorder by morethan one mechanism (i.e., tumor initiating cell reduction and disruptionof cellular adhesion) to provide additive or synergistic effects. Stillother preferred embodiments may take advantage of the cellularinternalization of ephrin-A ligands to deliver a modulator mediatedanti-cancer agent. In this regard it will be appreciated that thepresent invention is not limited by any particular mechanism of actionbut rather encompasses the broad use of the disclosed modulators totreat EFNA associated disorders (including various neoplasia).

Thus, another preferred embodiment of the invention comprises a methodof treating an EFNA associated disorder in a subject in need thereofcomprising the step of administering an EFNA modulator to said subject.In particularly preferred embodiments the EFNA modulator will beassociated (e.g., conjugated) with an anti-cancer agent. Moreover thebeneficial aspects of the instant invention, including any cellularadhesion disruption and collateral benefits, may be achieved whether thesubject tumor tissue exhibits elevated levels of EFNA or reduced ordepressed levels of EFNA as compared with normal adjacent tissue.

As alluded to above and discussed in more detail below there arecurrently six known ephrin-A ligands (i.e., EFNAs 1-6). In accordancewith the instant invention it will be appreciated that the disclosedmodulators may be generated, fabricated and/or selected to react with asingle ephrin-A ligand (e.g., EFNA4), a subset of ephrin-A ligands(e.g., EFNA4 and EFNA1) or all six ephrin-A ligands. More particularly,as described herein and set forth in the Examples below, preferredmodulators such as antibodies may be generated and selected so that theyreact or bind with domains or epitopes that are expressed on a singleephrin-A ligand or with epitopes that are conserved (at least to someextent) and presented across multiple or all EFNA polypeptides (e.g.,EFNAs 1 and 4 or EFNAs 3 and 4). This is significant with respect to theinstant invention in that, as shown in Example 18 below, certainephrin-A ligands have been found to be preferably expressed on TIC and,in combination, may serve as particularly effective therapeutic targetsthat provide for the selective reduction in tumorigenic cell frequencyand/or depletion of cancer stem cell populations.

Therefore, in a selected embodiment the invention comprises a pan-EFNAmodulator that immunospecifically associates with two or more ephrin-Aligands. In such embodiments the selected modulator may have beengenerated through immunization with a particular ligand (e.g., EFNA4)and associate or cross-react with the various subject ligands to agreater or lesser degree. Accordingly, in yet other embodiments thepresent invention comprises a method of treating a subject in needthereof comprising administering a therapeutically effective amount of apan-EFNA modulator. Still other embodiments comprise a method oftreating a subject in need thereof comprising administering atherapeutically effective amount of an EFNA modulator thatimmunospecifically associates with one or more ephrin-A ligands.

Accordingly, in yet other embodiments the present invention willcomprise a pan-EFNA modulator. In still other embodiments the presentinvention will comprise a method of treating an EFNA associated disorderin a subject in need thereof comprising the step of administering apan-EFNA modulator to said subject.

Of course it will be appreciated that the disclosed EFNA modulators maybe generated, fabricated and/or selected to preferentially react orassociate with a single ephrin-A ligand (e.g., EFNA4) and exhibitminimal or no association with any other ephrin-A ligand. Accordingly,other embodiments of the invention are directed to EFNA modulators thatimmunospecifically associate with a selected ephrin-A ligand and exhibitlittle or no association with any other ephrin-A ligand. In this regardpreferred embodiments disclosed herein will comprise methods of treatingan EFNA associated disorder in a subject in need thereof comprising thestep of administering an EFNA modulator wherein the EFNA modulatorimmunospecifically associates with a selected ephrin-A ligand and issubstantially non-reactive with any other ephrin-A ligand. Further,methods of generating, fabricating and selecting such modulators arewithin the scope of the instant invention.

Other facets of the instant invention exploit the ability of thedisclosed modulators to potentially disrupt cell adhesion interactionswhile simultaneously silencing tumor initiating cells. Such multi-activeEFNA modulators (e.g., EFNA antagonists) may prove to be particularlyeffective when used in combination with standard of care anti-canceragents or debulking agents. In addition, two or more EFNA antagonists(e.g. antibodies that specifically bind to two discrete epitopes on anephrin-A ligand or that associate with discrete ligands) may be used incombination in accordance with the present teachings. Moreover, asdiscussed in some detail below, the EFNA modulators of the presentinvention may be used in a conjugated or unconjugated state and,optionally, as a sensitizing agent in combination with a varietychemical or biological anti-cancer agents.

Thus, another preferred embodiment of the instant invention comprises amethod of sensitizing a tumor in a subject for treatment with ananti-cancer agent comprising the step of administering an EFNA modulatorto said subject. In a particularly preferred aspect of the invention theEFNA modulator will specifically result in a reduction of tumorinitiating cell frequency is as determined using in vitro or in vivolimiting dilution analysis thereby sensitizing the tumor for concomitantor subsequent debulking.

Similarly, as the compounds of the instant invention may exerttherapeutic benefits through various physiological mechanisms, thepresent invention is also directed to selected effectors or modulatorsthat are specifically fabricated to exploit certain cellular processes.For example, in certain embodiments the preferred modulator may beengineered to associate with EFNA on or near the surface of the tumorinitiating cell and stimulate the subject's immune response. In otherembodiments the modulator may comprise an antibody directed to anepitope that neutralizes ephrin-A ligand activity and interactions withephrin receptors which may impact adhesive and repulsive forces betweencells through integrin and cytoskeleton rearrangements. In yet otherembodiments the disclosed modulators may act by depleting or eliminatingthe EFNA associated cells. As such, it is important to appreciate thatthe present invention is not limited to any particular mode of actionbut rather encompasses any method or EFNA modulator that achieves thedesired outcome.

Within such a framework preferred embodiments of the disclosedembodiments are directed to a method of treating a subject sufferingfrom neoplastic disorder comprising the step of administering atherapeutically effective amount of at least one neutralizing EFNAmodulator.

Other embodiments are directed to a method of treating a subjectsuffering from an EFNA associated disorder comprising the step ofadministering a therapeutically effective amount of at least onedepleting EFNA modulator. A related method is directed to depleting EFNAassociated cells in a subject in need thereof comprising the step ofadministering an EFNA modulator.

In yet another embodiment the present invention provides methods ofmaintenance therapy wherein the disclosed effectors or modulators areadministered over a period of time following an initial procedure (e.g.,chemotherapeutic, radiation or surgery) designed to remove at least aportion of the tumor mass. Such therapeutic regimens may be administeredover a period of weeks, a period of months or even a period of yearswherein the EFNA modulators may act prophylactically to inhibitmetastasis and/or tumor recurrence. In yet other embodiments thedisclosed modulators may be administrated in concert with knowndebulking regimens to prevent or retard metastasis.

Beyond the therapeutic uses discussed above it will also be appreciatedthat the modulators of the instant invention may be used to diagnoseEFNA related disorders and, in particular, hyperproliferative disorders.In some embodiments the modulator may be administered to the subject anddetected or monitored in vivo. Those of skill in the art will appreciatethat such modulators may be labeled or associated with markers orreporters as disclosed below and detected using any one of a number ofstandard techniques (e.g., MRI or CAT scan). In other instances themodulators may be used in an in vitro diagnostic setting usingart-recognized procedures. As such, a preferred embodiment comprises amethod of diagnosing a hyperproliferative disorder in a subject in needthereof comprising the steps of:

-   -   a. obtaining a tissue sample from said subject;    -   b. contacting the tissue sample with at least one EFNA        modulator; and    -   c. detecting or quantifying the EFNA modulator associated with        the sample.

Such methods may be easily discerned in conjunction with the instantapplication and may be readily performed using generally availablecommercial technology such as automatic plate readers, dedicatedreporter systems, etc. In selected embodiments the EFNA modulator willbe associated with tumor perpetuating cells present in the sample. Inother preferred embodiments the detecting or quantifying step willcomprise a reduction of tumor initiating cell frequency and detectionthereof. Moreover, limiting dilution analysis may be conducted aspreviously alluded to above and will preferably employ the use ofPoisson distribution statistics to provide an accurate accounting as tothe reduction of frequency.

In a similar vein the present invention also provides kits that areuseful in the diagnosis and monitoring of EFNA associated disorders suchas cancer. To this end the present invention preferably provides anarticle of manufacture useful for diagnosing or treating EFNA associateddisorders comprising a receptacle comprising an EFNA modulator andinstructional materials for using said EFNA modulator to treat ordiagnose the EFNA associated disorder.

Other preferred embodiments of the invention also exploit the propertiesof the disclosed modulators as an instrument useful for identifying,isolating, sectioning or enriching populations or subpopulations oftumor initiating cells through methods such as fluorescence activatedcell sorting (FACS) or laser mediated sectioning.

As such, another preferred embodiment of the instant invention isdirected to a method of identifying, isolating, sectioning or enrichinga population of tumor initiating cells comprising the step of contactingsaid tumor initiating cells with an EFNA modulator.

The foregoing is a summary and thus contains, by necessity,simplifications, generalizations, and omissions of detail; consequently,those skilled in the art will appreciate that the summary isillustrative only and is not intended to be in any way limiting. Otheraspects, features, and advantages of the methods, compositions and/ordevices and/or other subject matter described herein will becomeapparent in the teachings set forth herein. The summary is provided tointroduce a selection of concepts in a simplified form that are furtherdescribed below in the Detailed Description. This summary is notintended to identify key features or essential features of the claimedsubject matter, nor is it intended to be used as an aid in determiningthe scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-C depict, respectively, the nucleic acid sequence encodinghuman EFNA4 (SEQ ID NO: 1), the corresponding amino acid sequence ofhuman EFNA4 isoform a (SEQ ID NO: 2) and an alignment of human EFNA4 a,b and c isoform sequences showing amino acid differences (SEQ ID NOS:2-4) whereas FIGS. 1 D-F depict, respectively, the nucleic acid sequenceencoding human EFNA1 (SEQ ID NO: 5), the corresponding amino acidsequence of human EFNA1 isoform a (SEQ ID NO: 6) and an alignment ofhuman EFNA1 a and b isoforms sequences showing amino acid differences(SEQ ID NOS: 6 and 7);

FIGS. 2A and 2B are graphical representations depicting the geneexpression levels of selected human ephrin-A ligands and ephrin-Areceptors in untreated (FIG. 2A) and in irinotecan treated (FIG. 2B)mice as measured using whole transcriptome sequencing of highly enrichedtumor progenitor cell (TProg) and tumor perpetuating cell (TPC) andnon-tumorigenic cell (NTG) populations obtained from a subset of wholecolorectal tumor specimens;

FIGS. 3A and 3B are graphical representations depicting the geneexpression levels of human ephrin-A4 ligand in colorectal tumor samples(FIG. 3A) and pancreatic tumor samples (FIG. 3B) as measured using wholetranscriptome sequencing of highly enriched tumor progenitor cell(TProg) and tumor perpetuating cell (TPC) and non-tumorigenic cell (NTG)populations or tumorigenic (TG) and non-tumorigenic cell (NTG)populations;

FIG. 4 is a graphical representation showing the relative geneexpression levels of human EFNA4 in highly enriched tumor progenitorcell (TProg) and tumor perpetuating cell (TPC) populations obtained frommice bearing one of four different non-traditional xenograft (NTX)colorectal or pancreatic tumor cell lines, and normalized againstnon-tumorigenic (NTG) enriched cell populations as measured usingquantitative RT-PCR;

FIGS. 5A and 5B are graphical representations showing the relative geneexpression levels of human EFNA4 as measured using RT-PCR in wholecolorectal tumor specimens from patients with Stage I-IV disease, asnormalized against the mean of expression in normal colon and rectumtissue (FIG. 5A) or matched with normal adjacent tissue (FIG. 5B);

FIGS. 6A-6E represent gene expression levels of human EFNA genes asmeasured for EFNA4 in FIGS. 6A and 6B by RT-PCR in whole tumor specimens(grey dot) or matched NAT (white dots) from patients with one ofeighteen different solid tumor types, in FIGS. 6C and 6D by RT-PCR forEFNA4 and EFNA1 in selected NTX tumor cell lines and by Western blotanalysis in FIG. 6E for EFNA4 in normal tissue and selected NTX tumorcell lines;

FIGS. 7A-7R depict the sequences of several EFNA modulators wherein FIG.7A is a tabular representation showing the genetic arrangement and theheavy and light chain CDR sequences (derived from VBASE2 analysis) ofdiscrete EFNA modulators isolated and cloned as described herein, FIGS.7B-7N provide murine heavy and light chain variable region nucleic acidand amino acid sequences for the same modulators set forth in FIG. 7Aand FIGS. 7O-7R provide heavy and light chain variable region nucleicacid and amino acid sequences of exemplary humanized versions ofdisclosed EFNA modulators;

FIGS. 8A-8D set forth biochemical and immunological properties ofexemplary modulators as represented in a tabular format in FIG. 8A, acomparison of the affinity of murine SC4.47 and humanized SC4.47respectively as determined using label free interaction analysis with afixed amount of antibody and serial dilutions of antigen in FIGS. 8B and8C and a tabular comparison of the properties of selected humanized andmurine modulators in FIG. 8D;

FIG. 9 illustrates cell surface binding properties of fifty exemplaryephrin-A ligand modulators of the instant invention with regard toJurkat E6 cells and Z138 cells respectively;

FIGS. 10A and 10B depict the binding of an ephrin-A ligand to cellsexpressing ephrin-A receptors in a dose dependent manner (FIG. 10A) andinhibition of ephrin-A ligand cell surface binding through exposure toexemplary disclosed modulators (FIG. 10B);

FIGS. 11A-11D are graphical representations illustrating the ability thedisclosed modulators to inhibit the cell surface binding of human andmurine ephrin-A ligand wherein FIG. 11A shows positive control curvesand FIGS. 11B-11D demonstrate the ability of three exemplary EFNAmodulators to reduce ligand binding;

FIGS. 12A-12E are graphical representations showing the ability of themodulators of the instant invention to inhibit the cell surface bindingof soluble ephrin-A receptor wherein FIG. 12A provides a standard curveof receptor binding, FIG. 12B illustrates the properties of exemplarymodulators as the concentration of the soluble receptor is varied, FIG.12C demonstrates the consequences of varying the concentration ofmodulator while holding the amount of receptor steady and FIGS. 12D and12E show the ability of the modulators to inhibit ephrin-A receptorbinding to ephrin-A4 and ephrin-A1 ligand, respectively;

FIGS. 13A-13C illustrate the ability of selected modulators of theinstant invention to cross-react with the mouse ortholog of ephrin-A4ligand wherein FIG. 13A illustrates a non-reactive modulator and FIG.13B and FIG. 13C illustrate murine and humanized modulators respectivelythat do cross-react;

FIGS. 14A-14D demonstrate the expression of ephrin-A ligand isupregulated in whole colorectal tumor samples (FIG. 14A) and in thetumorigenic subpopulation of colorectal NTX tumor cells (FIG. 14B) andin the tumorigenic subpopulation of a lung NTX cell line (FIG. 14D) butnot on normal peripheral blood mononuclear cells (FIG. 14C);

FIGS. 15A-15D illustrate the ability of selected modulators of theinstant invention to internalize upon binding with ephrin-A ligandswhere FIG. 15A shows the fluorescent shift associated with threeexemplary modulators, FIG. 15B demonstrates that nineteen disclosedmodulators exhibit a delta mean fluorescent intensity indicative ofinternalization, FIG. 15C shows relatively little internalization in lowEFNA expressing cells and FIG. 15D shows substantial internalizationwith respect to cells expressing high levels of EFNA;

FIGS. 16A-16F provide evidence that the disclosed modulators mayeffectively be used as targeting moieties to direct cytotoxic payloadsto cells expressing ephrin-A ligands in which the downward sloping curveis indicative of cell killing through internalized and wherein FIG. 16Ashows the killing effects of modulator SC4.5, FIG. 16B illustrates theability of selected modulators to internalize and kill lung and skin NTXtumor cell lines, FIGS. 16C and 16D show that modulators carry anassociated cytotoxin into HEK293T cells (FIG. 16C) and HEK-.hEFNA4 cells(FIG. 16D), FIG. 16E illustrates that humanized modulators reactsimilarly and FIG. 16F demonstrates killing of target cells expressingmouse or human ephrin-A ligand (note that throughout FIG. 16 themodulators may be termed E rather than SC4);

FIGS. 17A-17E are graphical representations of various aspects of abiochemical assay demonstrating the ability of the disclosed modulatorsto detect secreted ephrin-A ligand wherein FIG. 17A provides a standardcurve, FIG. 17B quantifies the level of secreted EFNA from selecthematologic tumors, FIG. 17C presents a correlation between tumor volumeand secreted EFNA, FIG. 17D establishes a range of circulating ephrin-Aligand in healthy adults and FIG. 17E demonstrates that patients withselected solid tumors have significantly higher levels of circulatingephrin-A ligand;

FIGS. 18A-18C are graphical representations illustrating that variousephrin-A ligand modulators can be used as targeting moieties toassociate cytotoxic payloads with selected cells in which the downwardsloping curve is indicative of cell killing through internalized toxinand wherein FIGS. 18A-18C specifically demonstrate the ability of themodulators SC4.2.1 (or E2.1) and SC9.65 (or 9M065) to mediate killing ofHEK293T cells overexpressing ephrin-A4 ligand (FIG. 18A), ephrin-A3ligand (FIG. 18B) and ephrin-Al ligand (FIG. 18C) in the presence ofbound Saporin;

FIGS. 19A and 19B illustrate the ability of ephrin-A ligands to interactselectively with numerous EPHA receptors wherein HEK293T cells only bindEPHA-ECD-Fc receptor constructs via endogenously expressed ephrin-Aligands to a limited degree (FIG. 19A) while HEK293T.hEFNA4 cells bindall tested EPHA receptor constructs to various degrees, except for EPHA1which does not bind (FIG. 19B); and

FIGS. 20A and 20B illustrate the ability of ephrin-A ligands to interactselectively with EPHB receptors wherein HEK293T cells only bindEPHB-ECD-Fc receptor constructs via endogenously expressed ephrin-Aligands to a limited degree (FIG. 20A) while HEK293T.hEFNA4 cells bindEphB2 but not EphB3 and EphB4 receptors (FIG. 20B).

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

While the present invention may be embodied in many different forms,disclosed herein are specific illustrative embodiments thereof thatexemplify the principles of the invention. It should be emphasized thatthe present invention is not limited to the specific embodimentsillustrated. Moreover, any section headings used herein are fororganizational purposes only and are not to be construed as limiting thesubject matter described.

As previously alluded to, it has surprisingly been found that theexpression of ephrin-A ligands (or EFNA) are associated with neoplasticgrowth and hyperproliferative disorders and that such ligands provideuseful tumor markers which may be exploited in the treatment of relateddiseases. More specifically, it has been discovered that EFNA modulatorssuch as those disclosed herein may advantageously be used in thediagnosis, theragnosis, treatment or prevention of neoplastic disordersin subjects in need thereof. Accordingly, while preferred embodiments ofthe invention will be discussed extensively below, particularly in thecontext of cancer stem cells and their interactions with the disclosedmodulators, those skilled in the art will appreciate that the scope ofthe instant invention is not limited by such exemplary embodiments.Rather, the present invention and the appended claims are broadly andexpressly directed to EFNA modulators and their use in the diagnosis,theragnosis, treatment or prevention of a variety of EFNA associated ormediated disorders, including neoplastic or hyperproliferativedisorders, regardless of any particular mechanism of action orspecifically targeted tumor component.

It will further be appreciated that, in contrast to many prior artdisclosures, the present invention is largely directed to ephrin ligandmodulators (i.e. EFN) rather than ephrin receptor (i.e. EPH) modulators.That is, while ephrin receptors have been widely implicated in severaltypes of disorders and generally targeted for therapeutic intervention,ephrin ligands have heretofore attracted much less attention. In partthis may be as a result of the promiscuous behavior attributed to theligands and the misplaced belief that such varied interactions made themuntenable therapeutic targets as pathway redundancy would likelycompensate for any ligand antagonism. However, as demonstrated hereinthe disclosed ephrin-A ligand modulators can effectively be used totarget and eliminate or otherwise incapacitate tumorigenic cells.Moreover, in selected embodiments the present invention comprisespan-EFNA modulators that associate or react with more than one eprhin-Aligand thereby providing an unexpected additive or synergistic effectthat may allow for quiescence of more than one ephrin ligand mediatedpathway.

Besides the general association discussed immediately above, theinventors have further discovered a heretofore unknown phenotypicalassociation between selected “tumor initiating cells” (TIC) and ephrin-Aligands. In this regard, it has been found that selected TICs expresselevated levels of ephrin-A ligands when compared to normal tissue andnon-tumorigenic cells (NTG), which together comprise much of a solidtumor. Thus, the ephrin-A ligands comprise tumor associated markers (orantigens) and have been found to provide effective agents for thedetection and suppression of TIC and associated neoplasia due toelevated levels of the proteins on cell surfaces or in the tumormicroenvironment. More specifically, it has further been discovered thatEFNA modulators, including immunoreactive antagonists and antibodiesthat associate or react with the proteins, effectively reduce thefrequency of tumor initiating cells and are therefore useful ineliminating, incapacitating, reducing, promoting the differentiation of,or otherwise precluding or limiting the ability of thesetumor-initiating cells to lie dormant and/or continue to fuel tumorgrowth, metastasis or recurrence in a patient. As discussed in moredetail below, the TIC tumor cell subpopulation is composed of both tumorperpetuating cells (TPC) and highly proliferative tumor progenitor cells(TProg).

In view of these discoveries, those skilled in the art will appreciatethat the present invention further provides EFNA modulators and theiruse in reducing the frequency of tumor initiating cells. As will bediscussed extensively below, EFNA modulators of the invention broadlycomprise any compound that recognizes, reacts, competes, antagonizes,interacts, binds, agonizes, or associates with an ephrin-A ligand or itsgene. By these interactions, the EFNA modulators thereby reduce ormoderate the frequency of tumor initiating cells. Exemplary modulatorsdisclosed herein comprise nucleotides, oligonucleotides,polynucleotides, peptides or polypeptides. In certain preferredembodiments the selected modulators will comprise antibodies to an EFNAor immunoreactive fragments or derivatives thereof. Such antibodies maybe antagonistic or agonistic in nature and may optionally be conjugatedor associated with a cytotoxic agent. In other embodiments, modulatorswithin the instant invention will comprise an EFNA construct comprisingan ephrin-A ligand or a reactive fragment thereof. It will beappreciated that such constructs may comprise fusion proteins and caninclude reactive domains from other polypeptides such as immunoglobulinsor biological response modifiers. In still other aspects, the EFNAmodulator will comprise a nucleic acid assembly that exerts the desiredeffects at a genomic level. Still other modulators compatible with theinstant teachings will be discussed in detail below.

Whichever form of modulator is ultimately selected it will preferably bein an isolated and purified state prior to introduction into a subject.In this regard the term “isolated EFNA modulator” shall be construed ina broad sense and in accordance with standard pharmaceutical practice tomean any preparation or composition comprising the modulator in a statesubstantially free of unwanted contaminants (biological or otherwise).As will be discussed in some detail below these preparations may bepurified and formulated as desired using various art recognizedtechniques. Of course, it will be appreciated that such “isolated”preparations may be intentionally formulated or combined with inert oractive ingredients as desired to improve the commercial, manufacturingor therapeutic aspects of the finished product and providepharmaceutical compositions.

II. EFNA Physiology

Ephrin receptor tyrosine kinases (EPH), type-I transmembrane proteins,comprise the largest family of receptor tyrosine kinases within animalgenomes and interact with ephrin ligands (EFN), which are also cellsurface associated. Receptors in the EPH subfamily typically have asingle kinase domain and an extracellular region containing a Cys-richdomain and 2 fibronectin type III repeats. Convention holds that ephrinreceptors are divided into two groups based on the similarity of theirextracellular domain sequences and their affinities for binding ephrin-Aand ephrin-B ligands. Previous research has shown that EPH mediatedsignaling events control multiple aspects of embryonic development,particularly in the nervous system and are important mediators ofcell-cell communication regulating cell attachment, shape, and mobility.Moreover, many members of the ephrin receptor family, as opposed toephrin ligands, have been identified as important markers and/orregulators of the development and progression of cancer. To date nineephrin-A receptors and six ephrin-B receptors are known

For the purposes of the instant application the terms “ephrin receptor,”“ephrin-A receptor,” “ephrin-B receptor,” “EPHA,” or “EPHB” (or EphA orEphB) may be used interchangeably and held to mean the specified family,subfamily or individual receptor (i.e., EPHA1, EPHA2, EPHA3, EPHA4,EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5,EPHB6) as dictated by context.

Based upon sequence analyses, ephrin ligands can be divided into twogroups: six ephrin-A ligands (or EFNA), typically anchored to the cellsurface via glycosylphosphatidylinositol linkages (although somenon-GPI-anchored proteins are produced through alternative splicing ofephrin mRNAs; e.g. EFNA4) and three ephrin-B ligands (or EFNB)containing a transmembrane domain and a short cytoplasmic region withconserved tyrosine residues and a PDZ-binding motif. EFNA ligandsinteract preferentially with any of the nine different EPHA receptors,whereas EFNB ligands interact preferentially with any of six differentEPHB receptors, although some specific EFNA-EPHB and EFNB-EPHAcross-interactions have been reported.

For the purposes of the instant application the terms “ephrin ligand,”“ephrin-A ligand,” “ephrin-B ligand,” “EFNA,” or “EFNB” may be usedinterchangeably and held to mean the specified family, subfamily orindividual receptor (i.e., EFNA1, EFNA2, EFNA3, EFNA4, EFNA5, EFNA6,EFNB1, EFNB2, EFNB3) as dictated by context. For example, the terms“ephrin-A4,” ephrin-A4 ligand” or “EFNA4” shall all be held to designatethe same family of protein isoforms (e.g., as set forth in FIG. 1C)while the terms “ephrin-A ligand” and “ENFA” shall be held to mean theephrin subfamily (i.e. A as opposed to B) comprising all six A typeligands and any isoforms thereof. In this regard an “ephrin-Amodulator,” “ephrin-A ligand modulator” or “EFNA modulator” means anymodulator (as defined herein) that associates, binds or reacts with oneor more A type ligand or isoform, or fragment or derivative thereof.

A more detailed summary of ephrin receptor and ligand nomenclature maybe found in Table 1 immediately below.

TABLE 1 Receptors new Ligands name previous names new name previousnames EphA1 Eph, Esk ephrin-A1 B61; LERK-1, EFL-1 EphA2 Eck, Myk2, Sek2ephrin-A2 ELF-1; Cek7-L, LERK-6 EphA3 Cek4, Mek4, Hek, Tyro4; ephrin-A3Ehk1-L, EFL-2, LERK-3 Hek4 EphA4 Sek, Sek1, Cek8, Hek8, ephrin-A4LERK-4; EFL-4 Tyro1 EphA5 Ehk1, Bsk, Cek7, Hek7; ephrin-A5 AL-1, RAGS;LERK-7, Rek7 EFL-5 EphA6 Ehk2; Hek12 ephrin-A6 EphA7 Mdk1, Hek11, Ehk3,Ebk, Cek11 EphA8 Eek; Hek3 EphA9 EphB1 Elk, Cek6, Net; Hek6 ephrin-B1LERK-2, Elk-L, EFL-3, Cek5-L; STRA1 EphB2 Cek5, Nuk, Erk, Qek5,ephrin-B2 Htk-L, ELF-2; LERK-5, Tyro5, Sek3; Hek5, Drt NLERK-1 EphB3Cek10, Hek2, Mdk5, ephrin-B3 NLERK-2, Elk-L3, Tyro6, Sek4 EFL-6, ELF-3;LERK-8 EphB4 Htk, Myk1, Tyro11; Mdk2 EphB5 Cek9; Hek9 EphB6 Mep

Eph Nomenclature Committee, Cell. 1997; 90 (3):403-4, which isincorporated herein in its entirety by reference.

As with all cell surface receptor-ligand interactions, engagement of theephrin receptor by an ephrin ligand ultimately results in the activationof intracellular signaling cascades. Although receptor-ligandinteractions may take place between molecules on the surface of the samecell (cis interactions), it is generally thought that cis interactionsdo not lead to the triggering of signaling cascades, or that cisinteractions may actually antagonize signaling cascades initiated bytrans interactions (e.g., between receptors and ligands on separatecells). One unique aspect of EPH-EFN trans interactions is the capacityfor the triggering of two signaling cascades upon receptor-ligandengagement—a forward signaling cascade in the cell expressing the ephrinreceptor, and a reverse signaling cascade in the cell expressing theephrin ligand. The activation of two separate signaling cascades mayreflect cell sorting and cell positioning processes that EPH and EFNhave evolved to co-ordinate in animal embryonic development.

EPH-EFN signaling frequently activates cell-signaling pathways thatregulate cytoskeletal dynamics and lead to modulation of the adhesiveand repulsive interactions between different types of cells. As ageneralization, EPH and EFN proteins are found at much higher levelsduring embryogenesis versus those observed in adult tissues, althoughcontinued low-level expression in the adult may reflect roles for thesemolecules in the normal function of tissues such as the adult gut, whichhas a well defined architecture arising from the migration ofdifferentiating cells from their source at the tissue stem cell in thecrypt to their final location at the surface of the villi facing theintestinal lumen. Since ephrin receptors were first identified inhepatocellular carcinomas, and EPH and EFN expression is typicallylimited in adults, reactivation of the expression of ephrin ligandsand/or ephrin receptors in human cancers may be linked to thededifferentiation of the cancer cells and/or the ability of these cancercells to invade surrounding normal tissue and to migrate from the siteof the primary tumor to distant locations. Other studies have suggestedthat EPH-EFN interactions also have a role in neoangiogenesis.

Consistent with findings that EPH-EFN interactions in non-lymphoidtissues regulate cellular interactions by generating adhesive orrepulsive forces between cells through integrin and cytoskeletonrearrangements, EPH and EFN molecules found on lymphoid cells have beenshown to mediate cell adhesion to extracellular matrix components,chemotaxis and cell migration. For example, EFNA1, (which binds to theEphA2 receptor and comprises, for example, an amino sequence as inGenbank accession NM_004428) engagement on primary CD4 and CD8 T cellshas been found to stimulate cell migration and enhance chemotaxis. LikeEFNA1, EFNA4 is expressed on primary CD4 T cells but, due to thepromiscuity of the EPH-EFN interaction, it is unclear if EFNA4engagement has similar effects on these cells. However, it has beendemonstrated that mature human B-lymphocytes express EFNA4 and secreteit upon activation. Further EFNA4, unlike any other EFN or EPH molecule,is also consistently expressed on or by B cells of chronic lymphocyticleukemia (CLL) patients. Interestingly, the expression of EFNA4 isoformsas measured by Q-PCR may be correlated with the clinical manifestationof the disease. Also, B cells from CLL patients known to have increasedexpression of EFNA4 showed impairment in transendothelial migrationpotential compared to B cells from healthy individuals. Apparentlyengagement of EFNA4 reduced the ability of CLL cells to adhere toextracellular matrix molecules and reduced their chemotactic response toCCL1. Together these reports suggest a role for EFNA4 in B and T celltrafficking and, when viewed in combination with the intracellularsignaling data discussed above, make ephrin-A ligands, and EFNA4 inparticular, very intriguing targets for the development of anti-cancertherapeutics.

In addition to the aforementioned characteristics the present disclosuredemonstrates that the expression of EFNA4 is elevated in various cancerstem cell populations. Along with concomitant upregulation of severalEPHA receptors in the bulk tumor, this raises the possibility that EFNA4mediated ligand receptor interactions may be triggering cell signalingcascades linked to tumor proliferation, neoangiogenesis and/or tumormetastasis. While not wishing to be bound by any particular theory it isbelieved that EFNA4 modulators of the present invention (particularlyantagonistic or neutralizing embodiments) act, at least in part, byeither reducing or eliminating tumor initiating cell frequency therebyinterfering with tumor propagation or survival in a different mannerthan traditional standard of care therapeutic regimens (e.g.irinotecan), or through immunotherapeutic signaling or delivering apayload able to kill EFNA4 expressing cells. For example, elimination ofTPC by antagonizing EFNA4 may include simply promoting cellproliferation in the face of chemotherapeutic regimens that eliminateproliferating cells, or promote differentiation of TPC such that theirself-renewal (i.e. unlimited proliferation and maintenance ofmultipotency) capacity is lost. Alternatively, in preferred embodimentsthe recruitment of cytotoxic T-cells to attack EFNA4 expressing cells,or delivery of a potent toxin conjugated to an anti-EFNA4 antibody thatis able to internalize, may selectively kill or otherwise incapacitateTPC.

As used herein the term EFNA4 (also known as ligand of eph-relatedkinase 4, LERK4; or eph-related receptor tyrosine kinase ligand 4,EFL-4) refers to naturally occurring human EFNA4 unless contextuallydictated otherwise. Representative EFNA4 protein orthologs include, butare not limited to, human (i.e. hEFNA4, NP_005218, NP_872631 orNP_872632), mouse (NP_031936), chimpanzee (XP_001153095, XP_001152971,XP_524893, and XP 001152916) and rat (NP_001101162). The transcribedhuman EFNA4 gene comprises at minimum 5817 bp from chromosome 1. ThreemRNA transcript variants have been described, each of which arises fromalternative splicing of the transcribed RNA: (1) a 1276 bp variant(NM_005227; EFNA4 transcript variant 1; SEQ ID NO: 1) which encodes a201 amino acid proprotein (NP_005218; EFNA4 variant a; SEQ ID NO: 2);(2) a 1110 bp variant (NM_182689; EFNA4 transcript variant 2) whichencodes a 207 amino acid proprotein (NM_872631; EFNA4 variant b; SEQ IDNO: 3); and (3) a 1111 bp variant (NM_182690; EFNA4 transcript variant3) which encodes a 193 amino acid proprotein (NP_872632; EFNA4 variantc; SEQ ID NO: 4). It will be appreciated that each of the human EFNA4proteins include a predicted signal or leader sequence comprising aminoacids 1-25 of SEQ ID NO: 2 which is clipped off to provide the matureform of the protein (i.e. 168-182 aa). This signal peptide targets thepolypeptide to the cell surface/secretory pathway. Due to thealternative splicing of the mRNA with consequent effects upon theprotein coding sequences, the protein isoforms are processed differentlyby the cell—isoform a is membrane localized and anchored to the cellsurface by a glycosylphosphatidylinositol (GPI) linkage, whereasisoforms b and c lack the GPI-anchor signal sequence and therefore areexpected to be secreted by the cell. An alignment of the three proteinisoforms of human EFNA4 is shown in FIG. 1C. As previously indicated,unless otherwise indicated by direct reference or contextual necessitythe term EFNA4 shall be directed to isoform a of human EFNA4 andimmunoreactive equivalents. It will further be appreciated that the termmay also refer to a derivative or fragment of a native or variant formof EFNA4 that contains an epitope to which an antibody or immunoreactivefragment can specifically bind.

III. Tumor Perpetuating Cells

In contrast to teachings of the prior art, the present inventionprovides EFNA modulators that are particularly useful for targetingtumor initiating cells, and especially tumor perpetuating cells, therebyfacilitating the treatment, management or prevention of neoplasticdisorders. More specifically, as previously indicated it hassurprisingly been found that specific tumor cell subpopulations expressEFNA and likely modify localized coordination of morphogen signalingimportant to cancer stem cell self-renewal and cell survival. Thus, inpreferred embodiments modulators of EFNA may be used to reduce tumorinitiating cell frequency in accordance with the present teachings andthereby facilitate the treatment or management of hyperproliferativediseases.

As used herein, the term tumor initiating cell (TIC) encompasses bothtumor perpetuating cells (TPC; i.e., cancer stem cells or CSC) andhighly proliferative tumor progenitor cells (termed TProg), whichtogether generally comprise a unique subpopulation (i.e. 0.1-40%) of abulk tumor or mass. For the purposes of the instant disclosure the termstumor perpetuating cells and cancer stem cells are equivalent and may beused interchangeably herein. Conversely, TPC differ from TProg in thatthey can completely recapitulate the composition of tumor cells existingwithin a tumor and have unlimited self-renewal capacity as demonstratedby serial transplantation (two or more passages through mice) of lownumbers of isolated cells. As will be discussed in more detail belowfluorescence-activated cell sorting (FACS) using appropriate cellsurface markers is a reliable method to isolate highly enriched cellsubpopulations (>99.5% purity) due, at least in part, to its ability todiscriminate between single cells and clumps of cells (i.e. doublets,etc.). Using such techniques it has been shown that when low cellnumbers of highly purified TProg cells are transplanted intoimmunocompromised mice they can fuel tumor growth in a primarytransplant. However, unlike purified TPC subpopulations the TProggenerated tumors do not completely reflect the parental tumor inphenotypic cell heterogeneity and are demonstrably inefficient atreinitiating serial tumorigenesis in subsequent transplants. Incontrast, TPC subpopulations completely reconstitute the cellularheterogeneity of parental tumors and can efficiently initiate tumorswhen serially isolated and transplanted. Thus, those skilled in the artwill recognize that a definitive difference between TPC and TProg,though both may be tumor generating in primary transplants, is theunique ability of TPC to perpetually fuel heterogeneous tumor growthupon serial transplantation at low cell numbers. Other common approachesto characterize TPC involve morphology and examination of cell surfacemarkers, transcriptional profile, and drug response although markerexpression may change with culture conditions and with cell line passagein vitro.

Accordingly, for the purposes of the instant invention tumorperpetuating cells, like normal stem cells that support cellularhierarchies in normal tissue, are preferably defined by their ability toself-renew indefinitely while maintaining the capacity for multilincagedifferentiation. Tumor perpetuating cells are thus capable of generatingboth tumorigenic progeny (i.e., tumor initiating cells: TPC and TProg)and non-tumorigenic (NTG) progeny. As used herein a non-tumorigenic cell(NTG) refers to a tumor cell that arises from tumor initiating cells,but does not itself have the capacity to self-renew or generate theheterogeneous lineages of tumor cells that comprise a tumor.Experimentally, NTG cells are incapable of reproducibly forming tumorsin mice, even when transplanted in excess cell numbers.

As indicated, TProg are also categorized as tumor initiating cells (orTIC) due to their limited ability to generate tumors in mice. TProg areprogeny of TPC and are typically capable of a finite number ofnon-self-renewing cell divisions. Moreover, TProg cells may further bedivided into early tumor progenitor cells (ETP) and late tumorprogenitor cells (LTP), each of which may be distinguished by phenotype(e.g., cell surface markers) and different capacities to recapitulatetumor cell architecture. In spite of such technical differences, bothETP and LTP differ functionally from TPC in that they are generally lesscapable of serially reconstituting tumors when transplanted at low cellnumbers and typically do not reflect the heterogeneity of the parentaltumor. Notwithstanding the foregoing distinctions, it has also beenshown that various TProg populations can, on rare occasion, gainself-renewal capabilities normally attributed to stem cells andthemselves become TPC (or CSC). In any event both types oftumor-initiating cells are likely represented in the typical tumor massof a single patient and are subject to treatment with the modulators asdisclosed herein. That is, the disclosed compositions are generallyeffective in reducing the frequency or altering the chemosensitivity ofsuch EFNA positive tumor initiating cells regardless of the particularembodiment or mix represented in a tumor.

In the context of the instant invention, TPC are more tumorigenic,relatively more quiescent and often more chemoresistant than the TProg(both ETP and LTP), NTG cells and the tumor-infiltrating non-TPC derivedcells (e.g., fibroblasts/stroma, endothelial & hematopoietic cells) thatcomprise the bulk of a tumor. Given that conventional therapies andregimens have, in large part, been designed to both debulk tumors andattack rapidly proliferating cells, TPC are likely to be more resistantto conventional therapies and regimens than the faster proliferatingTProg and other bulk tumor cell populations. Further, TPC often expressother characteristics that make them relatively chemoresistant toconventional therapies, such as increased expression of multi-drugresistance transporters, enhanced DNA repair mechanisms andanti-apoptotic proteins. These properties, each of which contribute todrug tolerance by TPC, constitute a key reason for the failure ofstandard oncology treatment regimens to ensure long-term benefit formost patients with advanced stage neoplasia; i.e. the failure toadequately target and eradicate those cells that fuel continued tumorgrowth and recurrence (i.e. TPC or CSC).

Unlike many of the aforementioned prior art treatments, the novelcompositions of the present invention preferably reduce the frequency oftumor initiating cells upon administration to a subject regardless ofthe form or specific target (e.g., genetic material, EFNA antibody orligand fusion construct) of the selected modulator. As noted above, thereduction in tumor initiating cell frequency may occur as a result of a)elimination, depletion, sensitization, silencing or inhibition of tumorinitiating cells; b) controlling the growth, expansion or recurrence oftumor initiating cells; c) interrupting the initiation, propagation,maintenance, or proliferation of tumor initiating cells; or d) byotherwise hindering the survival, regeneration and/or metastasis of thetumorigenic cells. In some embodiments, the reduction in the frequencyof tumor initiating cells occurs as a result of a change in one or morephysiological pathways. The change in the pathway, whether by reductionor elimination of the tumor initiating cells or by modifying theirpotential (e.g., induced differentiation, niche disruption) or otherwiseinterfering with their ability to exert affects on the tumor environmentor other cells, in turn allows for the more effective treatment ofEFNA-associated disorders by inhibiting tumorigenesis, tumor maintenanceand/or metastasis and recurrence.

Among the methods that can be used to assess such a reduction in thefrequency of tumor initiating cells is limiting dilution analysis eitherin vitro or in vivo, preferably followed by enumeration using Poissondistribution statistics or assessing the frequency of predefineddefinitive events such as the ability to generate tumors in vivo or not.While such limiting dilution analysis are the preferred methods ofcalculating reduction of tumor initiating cell frequency, other, lessdemanding methods, may also be used to effectively determine the desiredvalues, albeit slightly less accurately, and are entirely compatiblewith the teachings herein. Thus, as will be appreciated by those skilledin the art, it is also possible to determine reduction of frequencyvalues through well-known flow cytometric or immunohistochemical means.As to all the aforementioned methods see, for example, Dylla et al.2008, PMCID: PMC2413402 & Hoey et al. 2009, PMID: 19664991; each ofwhich is incorporated herein by reference in its entirety.

With respect to limiting dilution analysis, in vitro enumeration oftumor initiating cell frequency may be accomplished by depositing eitherfractionated or unfractionated human tumor cells (e.g. from treated anduntreated tumors, respectively) into in vitro growth conditions thatfoster colony formation. In this manner, colony forming cells might beenumerated by simple counting and characterization of colonies, or byanalysis consisting of, for example, the deposition of human tumor cellsinto plates in serial dilutions and scoring each well as either positiveor negative for colony formation at least 10 days after plating. In vivolimiting dilution experiments or analyses, which are generally moreaccurate in their ability to determine tumor initiating cell frequencyencompass the transplantation of human tumor cells, from eitheruntreated control or treated conditions, for example, intoimmunocompromised mice in serial dilutions and subsequently scoring eachmouse as either positive or negative for tumor formation at least 60days after transplant. The derivation of cell frequency values bylimiting dilution analysis in vitro or in vivo is preferably done byapplying Poisson distribution statistics to the known frequency ofpositive and negative events, thereby providing a frequency for eventsfulfilling the definition of a positive event; in this case, colony ortumor formation, respectively.

As to other methods compatible with the instant invention that may beused to calculate tumor initiating cell frequency, the most commoncomprise quantifiable flow cytometric techniques and immunohistochemicalstaining procedures. Though not as precise as the limiting dilutionanalysis techniques described immediately above, these procedures aremuch less labor intensive and provide reasonable values in a relativelyshort time frame. Thus, it will be appreciated that a skilled artisanmay use flow cytometric cell surface marker profile determinationemploying one or more antibodies or reagents that bind art recognizedcell surface proteins known to enrich for tumor initiating cells (e.g.,potentially compatible markers as are set forth in Example 1 below) andthereby measure TIC levels from various samples. In still anothercompatible method one skilled in the art might enumerate TIC frequencyin situ (e.g., in a tissue section) by immunohistochemistry using one ormore antibodies or reagents that are able to bind cell surface proteinsthought to demarcate these cells.

Using any of the above-referenced methods it is then possible toquantify the reduction in frequency of TIC (or the TPC therein) providedby the disclosed EFNA modulators (including those conjugated tocytotoxic agents) in accordance with the teachings herein. In someinstances, the compounds of the instant invention may reduce thefrequency of TIC (by a variety of mechanisms noted above, includingelimination, induced differentiation, niche disruption, silencing, etc.)by 10%, 15%, 20%, 25%, 30% or even by 35%. In other embodiments, thereduction in frequency of TIC may be on the order of 40%, 45%, 50%, 55%,60% or 65%. In certain embodiments, the disclosed compounds my reducethe frequency of TIC by 70%, 75%, 80%, 85%, 90% or even 95%. Of courseit will be appreciated that any reduction of the frequency of the TIClikely results in a corresponding reduction in the tumorigenicity,persistence, recurrence and aggressiveness of the neoplasia.

IV. EFNA Modulators

In any event, the present invention is directed to the use of EFNAmodulators, including EFNA antagonists, for the diagnosis, treatmentand/or prophylaxis of any one of a number of EFNA associatedmalignancies. The disclosed modulators may be used alone or inconjunction with a wide variety of anti-cancer compounds such aschemotherapeutic or immunotherapeutic agents or biological responsemodifiers. In other selected embodiments, two or more discrete EFNAmodulators may be used in combination to provide enhancedanti-neoplastic effects or may be used to fabricate multispecificconstructs.

In certain embodiments, the EFNA modulators of the present inventionwill comprise nucleotides, oligonucleotides, polynucleotides, peptidesor polypeptides. Even more preferably the modulators will comprisesoluble EFNA (sEFNA) or a form, variant, derivative or fragment thereofincluding, for example, EFNA fusion constructs (e.g., EFNA-Fc,EFNA-targeting moiety, etc.) or EFNA-conjugates (e.g., EFNA-PEG,EFNA-cytotoxic agent, EFNA-brm, etc.). It will also be appreciated that,in other embodiments, the EFNA modulators comprise antibodies (e.g.,anti-EFNA1 or anti-EFNA4 mAbs) or immunoreactive fragments orderivatives thereof. In particularly preferred embodiments themodulators of the instant invention will comprise neutralizingantibodies or derivatives or fragments thereof. In other embodiments theEFNA modulators may comprise internalizing antibodies or fragmentsthereof. In still other embodiments the EFNA modulators may comprisedepleting antibodies or fragments thereof. Moreover, as with theaforementioned fusion constructs, these antibody modulators may beconjugated, linked or otherwise associated with selected cytotoxicagents, polymers, biological response modifiers (BRMs) or the like toprovide directed immunotherapies with various (and optionally multiple)mechanisms of action. As alluded to above such antibodies may bepan-EFNA antibodies and associate with two or more ephrin-A ligands orimmunospecific antibodies that selectively react with one of the sixephrin-A ligands. In yet other embodiments the modulators may operate onthe genetic level and may comprise compounds as antisense constructs,siRNA, micro RNA and the like.

Based on the teachings herein, those skilled in the art will appreciatethat particularly preferred embodiments of the invention may comprisesEFNA4 or sEFNA1 or antibody modulators that associate with either, orboth, of EFNA4 or EFNA1.

It will further be appreciated that the disclosed EFNA modulators maydeplete, silence, neutralize, eliminate or inhibit growth, propagationor survival of tumor cells, particularly TPC, and/or associatedneoplasia through a variety of mechanisms, including agonizing orantagonizing selected pathways or eliminating specific cells depending,for example, on the form of EFNA modulator, any associated payload ordosing and method of delivery. Accordingly, while preferred embodimentsdisclosed herein are directed to the depletion, inhibition or silencingof specific tumor cell subpopulations such as tumor perpetuating cells,it must be emphasized that such embodiments are merely illustrative andnot limiting in any sense. Rather, as set forth in the appended claims,the present invention is broadly directed to EFNA modulators and theiruse in the treatment, management or prophylaxis of various EFNAassociated hyperproliferative disorders irrespective of any particularmechanism or target tumor cell population.

In the same sense disclosed embodiments of the instant invention maycomprise one or more EFNA antagonists. To that end it will beappreciated that EFNA antagonists of the instant invention may compriseany ligand, polypeptide, peptide, fusion protein, antibody orimmunologically active fragment or derivative thereof that recognizes,reacts, binds, combines, competes, associates or otherwise interactswith the EFNA protein or fragment thereof and eliminates, silences,reduces, inhibits, hinders, restrains or controls the growth of tumorinitiating cells or other neoplastic cells including bulk tumor or NTGcells. In selected embodiments the EFNA modulator comprises an EFNAantagonist.

As used herein an antagonist refers to a molecule capable ofneutralizing, blocking, inhibiting, abrogating, reducing or interferingwith the activities of a particular or specified protein, including thebinding of receptors to ligands or the interactions of enzymes withsubstrates. More generally antagonists of the invention may compriseantibodies and antigen-binding fragments or derivatives thereof,proteins, peptides, glycoproteins, glycopeptides, glycolipids,polysaccharides, oligosaccharides, nucleic acids, antisense constructs,siRNA, miRNA, bioorganic molecules, peptidomimetics, pharmacologicalagents and their metabolites, transcriptional and translation controlsequences, and the like. Antagonists may also include small moleculeinhibitors, fusion proteins, receptor molecules and derivatives whichbind specifically to the protein thereby sequestering its binding to itssubstrate target, antagonist variants of the protein, antisensemolecules directed to the protein, RNA aptamers, and ribozymes againstthe protein.

As used herein and applied to two or more molecules or compounds, theterms recognizes or associates shall be held to mean the reaction,binding, specific binding, combination, interaction, connection,linkage, uniting, coalescence, merger or joining, covalently ornon-covalently, of the molecules whereby one molecule exerts an effecton the other molecule.

Moreover, as demonstrated in the examples herein, some modulators ofhuman EFNA may, in certain cases, cross-react with EFNA from a speciesother than human (e.g., murine). In other cases exemplary modulators maybe specific for one or more isoforms of human EFNA and will not exhibitcross-reactivity with EFNA orthologs from other species. Of course, inconjunction with the teachings herein such embodiments may comprisepan-EFNA antibodies that associate with two or more ephrin-A ligandsfrom a single species or antibodies that exclusively associate with asingle ephrin-A ligand.

In any event, and as will be discussed in more detail below, thoseskilled in the art will appreciate that the disclosed modulators may beused in a conjugated or unconjugated form. That is, the modulator may beassociated with or conjugated to (e.g. covalently or non-covalently)pharmaceutically active compounds, biological response modifiers,anti-cancer agents, cytotoxic or cytostatic agents, diagnostic moietiesor biocompatible modifiers. In this respect it will be understood thatsuch conjugates may comprise peptides, polypeptides, proteins, fusionproteins, nucleic acid molecules, small molecules, mimetic agents,synthetic drugs, inorganic molecules, organic molecules andradioisotopes. Moreover, as indicated herein the selected conjugate maybe covalently or non-covalently linked to the EFNA modulator in variousmolar ratios depending, at least in part, on the method used to effectthe conjugation.

V. Antibodies

a. Overview

As previously alluded to particularly preferred embodiments of theinstant invention comprise EFNA modulators in the form of antibodies.The term antibody is used in the broadest sense and specifically coverssynthetic antibodies, monoclonal antibodies, oligoclonal or polyclonalantibodies, multiclonal antibodies, recombinantly produced antibodies,intrabodies, multispecific antibodies, bispecific antibodies, monovalentantibodies, multivalent antibodies, human antibodies, humanizedantibodies, chimeric antibodies, CDR-grafted antibodies, primatizedantibodies, Fab fragments, F(ab′) fragments, single-chain FvFcs(scFvFc), single-chain Fvs (scFv), anti-idiotypic (anti-Id) antibodiesand any other immunologically active antibody fragments so long as theyexhibit the desired biological activity (i.e., EFNA association orbinding). In a broader sense, the antibodies of the present inventioninclude immunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site, where these fragments may or may not be fused to anotherimmunoglobulin domain including, but not limited to, an Fc region orfragment thereof. Further, as outlined in more detail herein, the termsantibody and antibodies specifically include Fc variants as describedbelow, including full length antibodies and variant Fe-Fusionscomprising Fc regions, or fragments thereof, optionally comprising atleast one amino acid residue modification and fused to animmunologically active fragment of an immunoglobulin.

As discussed in more detail below, the generic terms antibody orimmunoglobulin comprises five distinct classes of antibody that can bedistinguished biochemically and, depending on the amino acid sequence ofthe constant domain of their heavy chains, can readily be assigned tothe appropriate class. For historical reasons, the major classes ofintact antibodies are termed IgA, IgD, IgE, IgG, and IgM. In humans, theIgG and IgA classes may be further divided into recognized subclasses(isotypes), i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 depending onstructure and certain biochemical properties. It will be appreciatedthat the IgG isotypes in humans are named in order of their abundance inserum with IgG1 being the most abundant.

While all five classes of antibodies (i.e. IgA, IgD, IgE, IgG, and IgM)and all isotypes (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), as wellas variations thereof, are within the scope of the present invention,preferred embodiments comprising the IgG class of immunoglobulin will bediscussed in some detail solely for the purposes of illustration. Itwill be understood that such disclosure is, however, merelydemonstrative of exemplary compositions and methods of practicing thepresent invention and not in any way limiting of the scope of theinvention or the claims appended hereto.

In this respect, human IgG immunoglobulins comprise two identical lightpolypeptide chains of molecular weight approximately 23,000 Daltons, andtwo identical heavy chains of molecular weight 53,000-70,000 dependingon the isotype. Heavy-chain constant domains that correspond to thedifferent classes of antibodies are denoted by the corresponding lowercase Greek letter α, δ, ϵ, γ, and μ, respectively. The light chains ofthe antibodies from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa (κ) and lambda (λ), based on theamino acid sequences of their constant domains. Those skilled in the artwill appreciate that the subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

The four chains are joined by disulfide bonds in a Y configurationwherein the light chains bracket the heavy chains starting at the mouthof the Y and continuing through the variable region to the dual ends ofthe Y. Each light chain is linked to a heavy chain by one covalentdisulfide bond while two disulfide linkages in the hinge region join theheavy chains. The respective heavy and light chains also have regularlyspaced intrachain disulfide bridges the number of which may vary basedon the isotype of IgG.

Each heavy chain has at one end a variable domain (V_(H)) followed by anumber of constant domains. Each light chain has a variable domain atone end (V_(L)) and a constant domain at its other end; the constantdomain of the light chain is aligned with the first constant domain ofthe heavy chain, and the light chain variable domain is aligned with thevariable domain of the heavy chain. In this regard, it will beappreciated that the variable domains of both the light (V_(L)) andheavy (V_(H)) chain portions determine antigen recognition andspecificity. Conversely, the constant domains of the light chain (C_(L))and the heavy chain (C_(H)1, C_(H)2 or C_(H)3) confer and regulateimportant biological properties such as secretion, transplacentalmobility, circulation half-life, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. Thus, the amino or N-terminus of the antibody comprisesthe variable region and the carboxy or C-terminus comprises the constantregion. Thus, the C_(H)3 and C_(L) domains actually comprise thecarboxy-terminus of the heavy and light chain, respectively.

The term variable refers to the fact that certain portions of thevariable domains differ extensively in sequence among immunoglobulinsand these hot spots largely define the binding and specificitycharacteristics of a particular antibody. These hypervariable sitesmanifest themselves in three segments, known as complementaritydetermining regions (CDRs), in both the light-chain and the heavy-chainvariable domains respectively. The more highly conserved portions ofvariable domains flanking the CDRs are termed framework regions (FRs).More specifically, in naturally occurring monomeric IgG antibodies, thesix CDRs present on each arm of the antibody are short, non-contiguoussequences of amino acids that are specifically positioned to form theantigen binding site as the antibody assumes its three dimensionalconfiguration in an aqueous environment.

The framework regions comprising the remainder of the heavy and lightvariable domains show less inter-molecular variability in amino acidsequence. Rather, the framework regions largely adopt a β-sheetconformation and the CDRs form loops which connect, and in some casesform part of, the β-sheet structure. Thus, these framework regions actto form a scaffold that provides for positioning the six CDRs in correctorientation by inter-chain, non-covalent interactions. Theantigen-binding site formed by the positioned CDRs defines a surfacecomplementary to the epitope on the immunoreactive antigen (i.e. EFNA4).This complementary surface promotes the non-covalent binding of theantibody to the immunoreactive antigen epitope. It will be appreciatedthat the position of CDRs can be readily identified by one of ordinaryskill in the art.

As discussed in more detail below and shown in the appended Examples,all or part of the heavy and light chain variable regions may berecombined or engineered using standard recombinant and expressiontechniques to provide effective antibodies. That is, the heavy or lightchain variable region from a first antibody (or any portion thereof) maybe mixed and matched with any selected portion of the heavy or lightchain variable region from a second antibody. For example, in oneembodiment, the entire light chain variable region comprising the threelight chain CDRs of a first antibody may be paired with the entire heavychain variable region comprising the three heavy chain CDRs of a secondantibody to provide an operative antibody. Moreover, in otherembodiments, individual heavy and light chain CDRs derived from variousantibodies may be mixed and matched to provide the desired antibodyhaving optimized characteristics. Thus, an exemplary antibody maycomprise three light chain CDRs from a first antibody, two heavy chainCDRs derived from a second antibody and a third heavy chain CDR from athird antibody.

More specifically, in the context of the instant invention it will beappreciated that any of the disclosed heavy and light chain CDRs in FIG.7A may be rearranged in this manner to provide optimized anti-EFNA (e.g.anti-hEFNA4) antibodies in accordance with the instant teachings. Thatis, one or more of the CDRs disclosed in FIG. 7A may be incorporated inan EFNA modulator and, in particularly preferred embodiments, in a CDRgrafted or humanized antibody that immunospecifically associates withone or more ephrin-A ligands.

In any event, the complementarity determining regions residue numbersmay be defined as those of Kabat et al. (1991, NIH Publication 91-3242,National Technical Information Service, Springfield, Va.), specifically,residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chainvariable domain and 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) in theheavy chain variable domain. Note that CDRs vary considerably fromantibody to antibody (and by definition will not exhibit homology withthe Kabat consensus sequences). Maximal alignment of framework residuesfrequently requires the insertion of spacer residues in the numberingsystem, to be used for the Fv region. In addition, the identity ofcertain individual residues at any given Kabat site number may vary fromantibody chain to antibody chain due to interspecies or allelicdivergence. See also Chothia et al., J. Mol. Biol. 196:901-917 (1987);Chothia et al., Nature 342, pp. 877-883 (1989) and by MacCallum et al.,J. Mol. Biol. 262:732-745 (1996) where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Each of the aforementioned references is incorporated herein byreference in its entirety and the amino acid residues which encompassCDRs as defined by each of the above cited references are set forth forcomparison.

CDR Definitions

Kabat¹ Chothia² MacCallum³ V_(H) CDR1 31-35 26-32 30-35 V_(H) CDR2 50-6553-55 47-58 V_(H) CDR3  95-102  96-101  93-101 V_(L) CDR1 24-34 26-3230-36 V_(L) CDR2 50-56 50-52 46-55 V_(L) CDR3 89-97 91-96 89-96 ¹Residuenumbering follows the nomenclature of Kabat et al., supra ²Residuenumbering follows the nomenclature of Chothia et al., supra ³Residuenumbering follows the nomenclature of MacCallum et al., supra

For purposes of convenience the CDRs set forth in FIG. 7A (SEQ ID NOS:8-59 and 70-95) were derived from VBASE2 analysis though given thecontent of the instant application one skilled in the art could readilyidentify and enumerate the CDRs as defined by Kabat et al. or MacCallumet al. for each respective heavy and light chain sequence. In thisregard CDRs as defined by Kabat et al. were used for the humanizationanalysis set forth in Example 7(b) and are underlined in FIGS. 7O-7R(SEQ ID NOS: 148-163) which depict humanized antibody sequences inaccordance with the instant invention. Accordingly, antibodiescomprising CDRs defined by all such nomenclature are expressly includedwithin the scope of the instant invention. More broadly the termvariable region CDR amino acid residue includes amino acids in a CDR asidentified using any sequence or structure based method as set forthabove.

As used herein the term variable region framework (FR) amino acidresidues refers to those amino acids in the framework region of an Igchain. The term framework region or FR region as used herein, includesthe amino acid residues that are part of the variable region, but arenot part of the CDRs (e.g., using the Kabat definition of CDRs).Therefore, a variable region framework is a non-contiguous sequencebetween about 100-120 amino acids in length but includes only thoseamino acids outside of the CDRs.

For the specific example of a heavy chain variable region and for theCDRs as defined by Kabat et al., framework region 1 corresponds to thedomain of the variable region encompassing amino acids 1-30; frameworkregion 2 corresponds to the domain of the variable region encompassingamino acids 36-49; framework region 3 corresponds to the domain of thevariable region encompassing amino acids 66-94, and framework region 4corresponds to the domain of the variable region from amino acids 103 tothe end of the variable region. The framework regions for the lightchain are similarly separated by each of the light claim variable regionCDRs. Similarly, using the definition of CDRs by Chothia et al. orMcCallum et al. the framework region boundaries are separated by therespective CDR termini as described above.

With the aforementioned structural considerations in mind, those skilledin the art will appreciate that the antibodies of the present inventionmay comprise any one of a number of functional embodiments. In thisrespect, compatible antibodies may comprise any immunoreactive antibody(as the term is defined herein) that provides the desired physiologicalresponse in a subject. While any of the disclosed antibodies may be usedin conjunction with the present teachings, certain embodiments of theinvention will comprise chimeric, humanized or human monoclonalantibodies or immunoreactive fragments thereof. Yet other embodimentsmay, for example, comprise homogeneous or heterogeneous multimericconstructs, Fc variants and conjugated or glycosylationally alteredantibodies. Moreover, it will be understood that such configurations arenot mutually exclusive and that compatible individual antibodies maycomprise one or more of the functional aspects disclosed herein. Forexample, a compatible antibody may comprise a single chain diabody withhumanized variable regions or a fully human full length IgG3 antibodywith Fc modifications that alter the glycosylation pattern to modulateserum half-life. Other exemplary embodiments are readily apparent tothose skilled in the art and may easily be discernable as being withinthe scope of the invention.

b. Antibody Generation

As is well known various host animals, including rabbits, mice, rats,etc. may be inoculated and used to provide antibodies in accordance withthe teachings herein. Art known adjuvants that may be used to increasethe immunological response, depending on the inoculated species include,but are not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants may protect the antigen from rapid dispersal bysequestering it in a local deposit, or they may contain substances thatstimulate the host to secrete factors that are chemotactic formacrophages and other components of the immune system. Preferably, if apolypeptide is being administered, the immunization schedule willinvolve two or more administrations of the polypeptide, spread out overseveral weeks.

After immunization of an animal with an EFNA immunogen (e.g., solubleEFNA4 or EFNA1) which may comprise selected isoforms and/or peptides, orlive cells or cell preparations expressing the desired protein,antibodies and/or antibody-producing cells can be obtained from theanimal using art recognized techniques. In some embodiments, polyclonalanti-EFNA antibody-containing serum is obtained by bleeding orsacrificing the animal. The serum may be used for research purposes inthe form obtained from the animal or, in the alternative, the anti-EFNAantibodies may be partially or fully purified to provide immunoglobulinfractions or homogeneous antibody preparations.

c. Monoclonal Antibodies

While polyclonal antibodies may be used in conjunction with certainaspects of the present invention, preferred embodiments comprise the useof EFNA reactive monoclonal antibodies. As used herein, the termmonoclonal antibody or mAb refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblemutations, e.g., naturally occurring mutations, that may be present inminor amounts. Thus, the modifier monoclonal indicates the character ofthe antibody as not being a mixture of discrete antibodies and may beused in conjunction with any type of antibody. In certain embodiments,such a monoclonal antibody includes an antibody comprising a polypeptidesequence that binds or associates with EFNA, wherein the EFNA-bindingpolypeptide sequence was obtained by a process that includes theselection of a single target binding polypeptide sequence from aplurality of polypeptide sequences.

In preferred embodiments, antibody-producing cell lines are preparedfrom cells isolated from the immunized animal. After immunization, theanimal is sacrificed and lymph node and/or splenic B cells areimmortalized by means well known in the art as shown in the appendedExamples). Methods of immortalizing cells include, but are not limitedto, transfecting them with oncogenes, infecting them with an oncogenicvirus and cultivating them under conditions that select for immortalizedcells, subjecting them to carcinogenic or mutating compounds, fusingthem with an immortalized cell, e.g., a myeloma cell, and inactivating atumor suppressor gene. If fusion with myeloma cells is used, the myelomacells preferably do not secrete immunoglobulin polypeptides (anon-secretory cell line). Immortalized cells are screened using anephrin-A ligand (including selected isoforms), or an immunoreactiveportion thereof. In a preferred embodiment, the initial screening isperformed using an enzyme-linked immunoassay (ELISA) or aradioimmunoassay.

More generally, discrete monoclonal antibodies consistent with thepresent invention can be prepared using a wide variety of techniquesknown in the art including hybridoma, recombinant techniques, phagedisplay technologies, yeast libraries, transgenic animals (e.g. aXenoMouse® or HuMAb Mouse®) or some combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniques such asbroadly described above and taught in more detail in Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) each of which is incorporatedherein. Using the disclosed protocols, antibodies are preferably raisedin mammals by multiple subcutaneous or intraperitoneal injections of therelevant antigen and an adjuvant. As previously discussed, thisimmunization generally elicits an immune response that comprisesproduction of antigen-reactive antibodies (that may be fully human ifthe immunized animal is transgenic) from activated splenocytes orlymphocytes. While the resulting antibodies may be harvested from theserum of the animal to provide polyclonal preparations, it is generallymore desirable to isolate individual lymphocytes from the spleen, lymphnodes or peripheral blood to provide homogenous preparations ofmonoclonal antibodies. Most typically, the lymphocytes are obtained fromthe spleen and immortalized to provide hybridomas.

For example, as described above, the selection process can be theselection of a unique clone from a plurality of clones, such as a poolof hybridoma clones, phage clones, or recombinant DNA clones. It shouldbe understood that a selected EFNA binding sequence can be furtheraltered, for example, to improve affinity for the target, to humanizethe target binding sequence, to improve its production in cell culture,to reduce its immunogenicity in vivo, to create a multispecificantibody, etc., and that an antibody comprising the altered targetbinding sequence is also a monoclonal antibody of this invention. Incontrast to polyclonal antibody preparations, which typically includediscrete antibodies directed against different determinants (epitopes),each monoclonal antibody of a monoclonal antibody preparation isdirected against a single determinant on an antigen. In addition totheir specificity, monoclonal antibody preparations are advantageous inthat they are typically uncontaminated by other immunoglobulins that maybe cross-reactive.

d. Chimeric Antibodies

In another embodiment, the antibody of the invention may comprisechimeric antibodies derived from covalently joined protein segments fromat least two different species or types of antibodies. It will beappreciated that, as used herein, the term chimeric antibodies isdirected to constructs in which a portion of the heavy and/or lightchain is identical with or homologous to corresponding sequences inantibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Inone exemplary embodiment, a chimeric antibody in accordance with theteachings herein may comprise murine V_(H) and V_(L) amino acidsequences and constant regions derived from human sources. In othercompatible embodiments a chimeric antibody of the present invention maycomprise a CDR grafted or humanized antibody as described below.

Generally, a goal of making a chimeric antibody is to create a chimerain which the number of amino acids from the intended subject species ismaximized. One example is the CDR-grafted antibody, in which theantibody comprises one or more complementarity determining regions(CDRs) from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the antibody chain(s) is/areidentical with or homologous to a corresponding sequence in antibodiesderived from another species or belonging to another antibody class orsubclass. For use in humans, the variable region or selected CDRs from arodent antibody often are grafted into a human antibody, replacing thenaturally occurring variable regions or CDRs of the human antibody.These constructs generally have the advantages of providing fullstrength modulator functions (e.g., CDC, ADCC, etc.) while reducingunwanted immune responses to the antibody by the subject.

e. Humanized Antibodies

Similar to the CDR grafted antibody is a humanized antibody. Generally,a humanized antibody is produced from a monoclonal antibody raisedinitially in a non-human animal. As used herein humanized forms ofnon-human (e.g., murine) antibodies are chimeric antibodies that containa minimal sequence derived from a non-human immunoglobulin. In oneembodiment, a humanized antibody is a human immunoglobulin (recipient oracceptor antibody) in which residues from a CDR of the recipientantibody are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, rabbit, or nonhuman primate havingthe desired specificity, affinity, and/or capacity.

Generally humanization of an antibody comprises an analysis of thesequence homology and canonical structures of both the donor andrecipient antibodies. In selected embodiments, the recipient antibodymay comprise consensus sequences. To create consensus human frameworks,frameworks from several human heavy chain or light chain amino acidsequences may be aligned to identify a consensus amino acid sequence.Moreover, in many instances, one or more framework residues in thevariable domain of the human immunoglobulin are replaced bycorresponding non-human residues from the donor antibody. Theseframework substitutions are identified by methods well known in the art,e.g., by modeling of the interactions of the CDR and framework residuesto identify framework residues important for antigen binding andsequence comparison to identify unusual framework residues at particularpositions. Such substitutions help maintain the appropriatethree-dimensional configuration of the grafted CDR(s) and often improveaffinity over similar constructs with no framework substitutions.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications may be made to further refine antibody performance usingwell-known techniques.

CDR grafting and humanized antibodies are described, for example, inU.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and5,530,101. In general, a humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDRs correspond to those of a non-humanimmunoglobulin, and all or substantially all of the framework regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally will also comprise at least a portion of an immunoglobulinconstant region (Fe), typically that of a human immunoglobulin. Forfurther details, see, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op.Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton,Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409. Stillanother method is termed humaneering and is described, for example, inU.S. 2005/0008625. For the purposes of the present application the termhumanized antibodies will be held to expressly include CDR graftedantibodies (i.e. human antibodies comprising one or more graftednon-human CDRs) with no or minimal framework substitutions.

Additionally, a non-human anti-EFNA antibody may also be modified byspecific deletion of human T cell epitopes or deimmunization by themethods disclosed in WO 98/52976 and WO 00/34317. Briefly, the heavy andlight chain variable regions of an antibody can be analyzed for peptidesthat bind to MHC Class II; these peptides represent potential T-cellepitopes (as defined in WO 98/52976 and WO 00/34317). For detection ofpotential T-cell epitopes, a computer modeling approach termed peptidethreading can be applied, and in addition a database of human MHC classII binding peptides can be searched for motifs present in the V_(H) andV_(L) sequences, as described in WO 98/52976 and WO 00/34317. Thesemotifs bind to any of the 18 major MHC class II DR allotypes, and thusconstitute potential T cell epitopes. Potential T-cell epitopes detectedcan be eliminated by substituting small numbers of amino acid residuesin the variable regions, or by single amino acid substitutions. As faras possible, conservative substitutions are made. Often, but notexclusively, an amino acid common to a position in human germlineantibody sequences may be used. After the deimmunizing changes areidentified, nucleic acids encoding V_(H) and V_(L) can be constructed bymutagenesis or other synthetic methods (e.g., de novo synthesis,cassette replacement, and so forth). A mutagenized variable sequencecan, optionally, be fused to a human constant region.

In selected embodiments, at least 60%, 65%, 70%, 75%, or 80% of thehumanized antibody variable region residues will correspond to those ofthe parental framework region (FR) and CDR sequences. In otherembodiments at least 85% or 90% of the humanized antibody residues willcorrespond to those of the parental framework region (FR) and CDRsequences. In a further preferred embodiment, greater than 95% of thehumanized antibody residues will correspond to those of the parentalframework region (FR) and CDR sequences.

Humanized antibodies may be fabricated using common molecular biologyand biomolecular engineering techniques as described herein. Thesemethods include isolating, manipulating, and expressing nucleic acidsequences that encode all or part of immunoglobulin Fv variable regionsfrom at least one of a heavy or light chain. Sources of such nucleicacid are well known to those skilled in the art and, for example, may beobtained from a hybridoma, eukaryotic cell or phage producing anantibody or immunoreactive fragment against a predetermined target, asdescribed above, from germline immunoglobulin genes, or from syntheticconstructs. The recombinant DNA encoding the humanized antibody can thenbe cloned into an appropriate expression vector.

Human germline sequences, for example, are disclosed in Tomlinson, I. A.et al. (1992) J. Mol. Biol. 227:776-798; Cook, G. P. et al. (1995)Immunol. Today 16: 237-242; Chothia, D. et al. (1992) J. Mol. Bio.227:799-817; and Tomlinson et al. (1995) EMBO J 14:4628-4638. The V BASEdirectory provides a comprehensive directory of human immunoglobulinvariable region sequences (See Retter et al., (2005) Nuc Acid Res 33:671-674). These sequences can be used as a source of human sequence,e.g., for framework regions and CDRs. As set forth herein consensushuman framework regions can also be used, e.g., as described in U.S.Pat. No. 6,300,064.

f. Human Antibodies

In addition to the aforementioned antibodies, those skilled in the artwill appreciate that the antibodies of the present invention maycomprise fully human antibodies. For the purposes of the instantapplication the term human antibody comprises an antibody whichpossesses an amino acid sequence that corresponds to that of an antibodyproduced by a human and/or has been made using any of the techniques formaking human antibodies as disclosed herein. This definition of a humanantibody specifically excludes a humanized antibody comprising non-humanantigen-binding residues.

Human antibodies can be produced using various techniques known in theart. As alluded to above, phage display techniques may be used toprovide immunoactive binding regions in accordance with the presentteachings. Thus, certain embodiments of the invention provide methodsfor producing anti-EFNA antibodies or antigen-binding portions thereofcomprising the steps of synthesizing a library of (preferably human)antibodies on phage, screening the library with a selected EFNA or anantibody-binding portion thereof, isolating phage that binds EFNA, andobtaining the immunoreactive fragments from the phage. By way ofexample, one method for preparing the library of antibodies for use inphage display techniques comprises the steps of immunizing a non-humananimal comprising human or non-human immunoglobulin loci with theselected EFNA or an antigenic portion thereof to create an immuneresponse, extracting antibody-producing cells from the immunized animal;isolating RNA encoding heavy and light chains of antibodies of theinvention from the extracted cells, reverse transcribing the RNA toproduce cDNA, amplifying the cDNA using primers, and inserting the cDNAinto a phage display vector such that antibodies are expressed on thephage. More particularly, DNA encoding the V_(H) and V_(L) domains arerecombined together with an scFv linker by PCR and cloned into aphagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector may thenbe electroporated in E. coli and then the E. coli is infected withhelper phage. Phage used in these methods are typically filamentousphage including fd and M13 and the V_(H) and V_(L) domains are usuallyrecombinantly fused to either the phage gene III or gene VIII.

Recombinant human anti-EFNA antibodies of the invention may be isolatedby screening a recombinant combinatorial antibody library prepared asabove. In a preferred embodiment, the library is a scFv phage displaylibrary, generated using human V_(L) and V_(H) cDNAs prepared from mRNAisolated from B cells. Methods for preparing and screening suchlibraries are well known in the art and kits for generating phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, catalog no. 27-9400-01; and theStratagene SurfZAP™ phage display kit, catalog no. 240612). There alsoare other methods and reagents that can be used in generating andscreening antibody display libraries (see, e.g., U.S. Pat. No.5,223,409; PCT Publication Nos. WO 92/18619, WO 91/17271, WO 92/20791,WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al.,Bio/Technology 9:1370-1372 (1991); Hay et al., Hum. Antibod. Hybridomas3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989); McCafferty etal., Nature 348:552-554 (1990); Griffiths et al., EMBO J. 12:725-734(1993); Hawkins et al., J. Mol. Biol. 226:889-896 (1992); Clackson etal., Nature 352:624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA89:3576-3580 (1992); Garrad et al., Bio/Technology 9:1373-1377 (1991);Hoogenboom et al., Nuc. Acid Res. 19:4133-4137 (1991); and Barbas etal., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(a) of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in the art. Forexample, mutation can be introduced at random in vitro by usingerror-prone polymerase (reported in Leung et al., Technique, 1: 11-15(1989)) in the method of Hawkins et al., J. Mol. Biol., 226: 889-896(1992) or in the method of Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992). Additionally, affinity maturation can be performed byrandomly mutating one or more CDRs, e.g. using PCR with primers carryingrandom sequence spanning the CDR of interest, in selected individual Fvclones and screening for higher affinity clones. WO 9607754 described amethod for inducing mutagenesis in a complementarity determining regionof an immunoglobulin light chain to create a library of light chaingenes. Another effective approach is to recombine the V_(H) or V_(L)domains selected by phage display with repertoires of naturallyoccurring V domain variants obtained from unimmunized donors and screenfor higher affinity in several rounds of chain reshuffling as describedin Marks et al., Biotechnol., 10: 779-783 (1992). This technique allowsthe production of antibodies and antibody fragments with a dissociationconstant K_(d) (k_(off)/k_(on)) of about 10⁻⁹ M or less.

It will further be appreciated that similar procedures may be employedusing libraries comprising eukaryotic cells (e.g., yeast) that expressbinding pairs on their surface. As with phage display technology, theeukaryotic libraries are screened against the antigen of interest (i.e.,EFNA) and cells expressing candidate-binding pairs are isolated andcloned. Steps may be taken to optimize library content and for affinitymaturation of the reactive binding pairs. See, for example, U.S. Pat.No. 7,700,302 and U.S. Ser. No. 12/404,059. In one embodiment, the humanantibody is selected from a phage library, where that phage libraryexpresses human antibodies (Vaughan et al. Nature Biotechnology14:309-314 (1996): Sheets et al. Proc. Natl. Acad. Sci. 95:6157-6162(1998)); Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks etal., J. Mol. Biol, 222:581 (1991)). In other embodiments human bindingpairs may be isolated from combinatorial antibody libraries generated ineukaryotic cells such as yeast. See e.g., U.S. Pat. No. 7,700,302. Suchtechniques advantageously allow for the screening of large numbers ofcandidate modulators and provide for relatively easy manipulation ofcandidate sequences (e.g., by affinity maturation or recombinantshuffling).

Human antibodies can also be made by introducing human immunoglobulinloci into transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and U.S. Pat. Nos. 6,075,181and 6,150,584 regarding Xenomouse® technology along with the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368:812-13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51(1996); Neuberger, Nature Biotechnology 14: 826 (1996); Lonberg andHuszar, Intern. Rev. Immunol. 13:65-93 (1995). Alternatively, the humanantibody may be prepared via immortalization of human B-lymphocytesproducing an antibody directed against a target antigen (such Blymphocytes may be recovered from an individual suffering from aneoplastic disorder or may have been immunized in vitro). See, e.g.,Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J. Immunol, 147 (1):86-95 (1991); and U.S.Pat. No. 5,750,373.

VI. Antibody Characteristics

No matter how obtained or which of the aforementioned forms the antibodymodulator takes (e.g., humanized, human, etc.) the preferred embodimentsof the disclosed modulators may exhibit various characteristics. In thisregard anti-EFNA antibody-producing cells (e.g., hybridomas or yeastcolonies) may be selected, cloned and further screened for desirablecharacteristics including, for example, robust growth, high antibodyproduction and, as discussed in more detail below, desirable antibodycharacteristics. Hybridomas can be expanded in vivo in syngeneicanimals, in animals that lack an immune system, e.g., nude mice, or incell culture in vitro. Methods of selecting, cloning and expandinghybridomas and/or colonies, each of which produces a discrete antibodyspecies, are well known to those of ordinary skill in the art.

a. Neutralizing Antibodies

In particularly preferred embodiments the modulators of the instantinvention will comprise neutralizing antibodies or derivative orfragment thereof. The term neutralizing antibody or neutralizingantagonist refers to an antibody or antagonist that binds to orinteracts with an ephrin-A ligand and prevents binding or association ofthe ligand to its binding partner (e.g., EPHA receptor) therebyinterrupting the biological response that otherwise would result fromthe interaction of the molecules. In assessing the binding andspecificity of an antibody or immunologically functional fragment orderivative thereof, an antibody or fragment will substantially inhibitbinding of the ligand to its binding partner or substrate when an excessof antibody reduces the quantity of binding partner bound to the targetmolecule by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%,95%, 97%, 99% or more as measured, for example, in an in vitrocompetitive binding assay (see e.g., Examples 9-12 herein). In the caseof antibodies to EFNA4 for example, a neutralizing antibody orantagonist will preferably diminish the ability of EFNA4 to bind toEphA4 by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%,95%, 97%, 99% or more. It will be appreciated that this diminishedactivity may be measured directly using art recognized techniques or maybe measured by the impact such reduction will have on EPH (e.g., EPHA4)receptor activity.

b. Internalizing Antibodies

While evidence indicates that selected ephrin-A ligands or theirisoforms may be present in a soluble form, at least some EFNA (e.g.,EFNA1 and EFNA4) likely remains associated with the cell surface therebyallowing for internalization of the disclosed modulators. Accordingly,the anti-EFNA antibodies of the instant invention may be internalized,at least to some extent, by cells that express an ephrin-A ligand. Forexample, an anti-EFNA4 antibody that binds to EFNA4 on the surface of atumor-initiating cell may be internalized by the tumor-initiating cell.In particularly preferred embodiments such anti-EFNA antibodies may beassociated with or conjugated to anti-cancer agents such as cytotoxicmoieties that kill the cell upon internalization.

As used herein, an anti-EFNA antibody that internalizes is one that istaken up by the cell upon binding to an EFNA associated with a mammaliancell. The internalizing antibody includes antibody fragments, human orhumanized antibody and antibody conjugates. Internalization may occur invitro or in vivo. For therapeutic applications, internalization mayoccur in vivo. The number of antibody molecules internalized may besufficient or adequate to kill an EFNA-expressing cell, especially anEFNA-expressing tumor initiating cell. Depending on the potency of theantibody or antibody conjugate, in some instances, the uptake of asingle antibody molecule into the cell is sufficient to kill the targetcell to which the antibody binds. For example, certain toxins are highlypotent in killing such that internalization of one molecule of the toxinconjugated to the antibody is sufficient to kill the tumor cell. Whetheran anti-EFNA antibody internalizes upon binding EFNA on a mammalian cellcan be determined by various assays including those described in theExamples below (e.g., Examples 15 and 16). Methods of detecting whetheran antibody internalizes into a cell are also described in U.S. Pat. No.7,619,068 which is incorporated herein by reference in its entirety.

c. Depleting Antibodies

In other preferred embodiments the modulators of the instant inventionwill comprise depleting antibodies or derivatives or fragments thereof.The term depleting antibody refers to an antibody or fragment that bindsto or associates with an EFNA on or near the cell surface and induces,promotes or causes the death or elimination of the cell (e.g., bycomplement-dependent cytotoxicity or antibody-dependent cellularcytotoxicity). In some embodiments discussed more fully below theselected depleting antibodies will be associated or conjugated to acytotoxic agent. Preferably a depleting antibody will be able to remove,eliminate or kill at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%,95%, 97%, or 99% of tumor perpetuating cells in a defined cellpopulation. In some embodiments the cell population may compriseenriched, sectioned, purified or isolated tumor perpetuating cells. Inother embodiments the cell population may comprise whole tumor samplesor heterogeneous tumor extracts that comprise tumor perpetuating cells.Those skilled in the art will appreciate that standard biochemicaltechniques as described in the Examples below (e.g., Example 16) may beused to monitor and quantify the depletion of tumorigenic cells or tumorperpetuating cells in accordance with the teachings herein.

d. Epitope Binding

It will further be appreciated the disclosed anti-EFNA antibodies willassociate with, or bind to, discrete epitopes or determinants presentedby the selected target(s). As used herein the term epitope refers tothat portion of the target antigen capable of being recognized andspecifically bound by a particular antibody. When the antigen is apolypeptide such as EFNA, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation. More specifically, the skilled artisanwill appreciate the term epitope includes any protein determinantcapable of specific binding to an immunoglobulin or T-cell receptor orotherwise interacting with a molecule. Epitopic determinants generallyconsist of chemically active surface groupings of molecules such asamino acids or carbohydrate or sugar side chains and generally havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Additionally an epitope may be linearor conformational. In a linear epitope, all of the points of interactionbetween the protein and the interacting molecule (such as an antibody)occur linearly along the primary amino acid sequence of the protein. Ina conformational epitope, the points of interaction occur across aminoacid residues on the protein that are linearly separated from oneanother.

Once a desired epitope on an antigen is determined, it is possible togenerate antibodies to that epitope, e.g., by immunizing with a peptidecomprising the epitope using techniques described in the presentinvention. Alternatively, during the discovery process, the generationand characterization of antibodies may elucidate information aboutdesirable epitopes. From this information, it is then possible tocompetitively screen antibodies for binding to the same epitope. Anapproach to achieve this is to conduct competition studies to findantibodies that competitively bind with one another, i.e. the antibodiescompete for binding to the antigen. A high throughput process forbinning antibodies based upon their cross-competition is described in WO03/48731.

As used herein, the term binning refers to a method to group antibodiesbased on their antigen binding characteristics. The assignment of binsis somewhat arbitrary, depending on how different the observed bindingpatterns of the antibodies tested. Thus, while the technique is a usefultool for categorizing antibodies of the instant invention, the bins donot always directly correlate with epitopes and such initialdeterminations should be further confirmed by other art recognizedmethodology.

With this caveat one can determine whether a selected primary antibody(or fragment thereof) binds to the same epitope or cross competes forbinding with a second antibody by using methods known in the art and setforth in the Examples herein. In one embodiment, one allows the primaryantibody of the invention to bind to EFNA under saturating conditionsand then measures the ability of the secondary antibody to bind to EFNA.If the test antibody is able to bind to EFNA at the same time as theprimary anti-EFNA antibody, then the secondary antibody binds to adifferent epitope than the primary antibody. However, if the secondaryantibody is not able to bind to EFNA at the same time, then thesecondary antibody binds to the same epitope, an overlapping epitope, oran epitope that is in close proximity to the epitope bound by theprimary antibody. As known in the art and detailed in the Examplesbelow, the desired data can be obtained using solid phase direct orindirect radioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay, a Biacore™ system (i.e.,surface plasmon resonance—GE Healthcare), a ForteBio® Analyzer (i.e.,bio-layer interferometry—ForteBio, Inc.) or flow cytometric methodology.The term surface plasmon resonance, as used herein, refers to an opticalphenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix. In a particularly preferred embodiment, theanalysis is performed using a Biacore or ForteBio instrument asdemonstrated in the Examples below.

The term compete when used in the context of antibodies that compete forthe same epitope means competition between antibodies is determined byan assay in which the antibody or immunologically functional fragmentunder test prevents or inhibits specific binding of a reference antibodyto a common antigen. Typically, such an assay involves the use ofpurified antigen bound to a solid surface or cells bearing either ofthese, an unlabeled test immunoglobulin and a labeled referenceimmunoglobulin. Competitive inhibition is measured by determining theamount of label bound to the solid surface or cells in the presence ofthe test immunoglobulin. Usually the test immunoglobulin is present inexcess. Antibodies identified by competition assay (competingantibodies) include antibodies binding to the same epitope as thereference antibody and antibodies binding to an adjacent epitopesufficiently proximal to the epitope bound by the reference antibody forsteric hindrance to occur. Additional details regarding methods fordetermining competitive binding are provided in the Examples herein.Usually, when a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to a common antigen by at least40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, binding isinhibited by at least 80%, 85%, 90%, 95%, or 97% or more.

Besides epitope specificity the disclosed antibodies may becharacterized using a number of different physical characteristicsincluding, for example, binding affinities, melting temperature (Tm),and isoelectric points.

e. Binding Affinity

In this respect, the present invention further encompasses the use ofantibodies that have a high binding affinity for a selected EFNA or, inthe case of pan-antibodies, more than one type of ephrin-A ligand. Anantibody of the invention is said to specifically bind its targetantigen when the dissociation constant K_(d) (k_(off)/k_(on)) is ≤10⁻⁸M.The antibody specifically binds antigen with high affinity when theK_(d) is ≤5×10⁻⁹M, and with very high affinity when the K_(d) is≤5×10⁻¹⁰M. In one embodiment of the invention, the antibody has a K_(d)of ≤10⁻⁹M and an off-rate of about 1×10⁻⁴/sec. In one embodiment of theinvention, the off-rate is <1×10⁻⁵/sec. In other embodiments of theinvention, the antibodies will bind to EFNA with a K_(d) of betweenabout 10⁻⁸M and 10⁻¹⁰M, and in yet another embodiment it will bind witha K_(d)≤2×10⁻¹⁰M. Still other selected embodiments of the presentinvention comprise antibodies that have a disassociation constant orK_(d) (K_(off)/k_(on)) of less than 10⁻²M, less than 5×10⁻²M, less than10⁻³M, less than 5×10⁻³M, less than 10⁻⁴M, less than 5×10⁻⁴M, less than10⁻⁵M, less than 5×10⁻⁵M, less than 10⁻⁶M, less than 5×10⁻⁶M, less than10⁻⁷M, less than 5×10⁻⁷M, less than 10⁻⁸M, less than 5×10⁻⁸M, less than10⁻⁹M, less than 5×10⁻⁹M, less than 10⁻¹⁰M, less than 5×10⁻¹⁰M, lessthan 10⁻¹¹M, less than 5×10⁻¹¹M, less than 10⁻¹²M, less than 5×10⁻¹²M,less than 10⁻¹³M, less than 5×10⁻¹³M, less than 10⁻¹⁴M, less than5×10⁻¹⁴M, less than 10⁻¹⁵M or less than 5×10⁻¹⁵M.

In specific embodiments, an antibody of the invention thatimmunospecifically binds to EFNA has an association rate constant ork_(off) rate (EFNA (Ab)+antigen (Ag)^(k) _(on)←Ab-Ag) of at least10⁵M⁻¹s⁻¹, at least 2×10⁵M⁻¹s⁻¹, at least 5×10⁵M⁻¹s⁻¹, at least10⁶M⁻¹s⁻¹, at least 5×10⁶M⁻¹s⁻¹, at least 10⁷M⁻¹s⁻¹, at least5×10⁷M⁻¹s⁻¹, or at least 10⁸M⁻¹s⁻¹.

In another embodiment, an antibody of the invention thatimmunospecifically binds to EFNA has a k_(off) rate (EFNA (Ab)+antigen(Ag)^(k) _(off)←Ab-Ag) of less than 10⁻¹s⁻¹, less than 5×10⁻¹s⁻¹, lessthan 10⁻²s⁻¹, less than 5×10⁻²s⁻¹, less than 10⁻³s⁻¹, less than5×10⁻³s⁻¹, less than 10⁻⁴s⁻¹, less than 5×10⁴s⁻¹, less than 10⁻⁵s⁻¹,less than 5×10⁻⁵s⁻¹, less than 10⁻⁶s⁻¹, less than 5×10⁻⁶s⁻¹ less than10⁻⁷s⁻¹, less than 5×10⁻⁷s⁻¹, less than 10⁻⁸s⁻¹, less than 5×10⁻⁸s⁻¹,less than 10⁻⁹s⁻¹, less than 5×10⁻⁹s⁻¹ or less than 10⁻¹⁰ s⁻¹.

In other selected embodiments of the present invention anti-EFNAantibodies will have an affinity constant or K_(a) (k_(on)/k_(off)) ofat least 10²M⁻¹, at least 5×10²M⁻¹, at least 10³M⁻¹, at least 5×10³M⁻¹,at least 10⁴M⁻¹, at least 5×10⁴M⁻¹, at least 10⁵M⁻¹, at least 5×10⁵M⁻¹,at least 10⁶M⁻¹, at least 5×10⁶M⁻¹, at least 10⁷M⁻¹, at least 5×10⁷M⁻¹,at least 10⁸M⁻¹, at least 5×10⁸M⁻¹, at least 10⁹M⁻¹, at least 5×10⁹M⁻¹,at least 10¹⁰M⁻¹, at least 5×10¹⁰ M⁻¹, at least 10¹¹M⁻¹, at least5×10¹¹M⁻¹, at least 10¹²M⁻¹, at least 5×10¹²M⁻¹, at least 10¹³M⁻¹, atleast 5×10¹³M⁻¹, at least 10¹⁴M⁻¹, at least 5×10¹⁴M⁻¹, at least 10¹⁵M⁻¹or at least 5×10¹⁵M⁻¹.

f. Isoelectric Points

In addition to the aforementioned binding properties, anti-EFNAantibodies and fragments thereof, like all polypeptides, have anIsoelectric Point (pI), which is generally defined as the pH at which apolypeptide carries no net charge. It is known in the art that proteinsolubility is typically lowest when the pH of the solution is equal tothe isoelectric point (pI) of the protein. Therefore it is possible tooptimize solubility by altering the number and location of ionizableresidues in the antibody to adjust the pI. For example the pI of apolypeptide can be manipulated by making the appropriate amino acidsubstitutions (e.g., by substituting a charged amino acid such as alysine, for an uncharged residue such as alanine). Without wishing to bebound by any particular theory, amino acid substitutions of an antibodythat result in changes of the pI of said antibody may improve solubilityand/or the stability of the antibody. One skilled in the art wouldunderstand which amino acid substitutions would be most appropriate fora particular antibody to achieve a desired pI.

The pI of a protein may be determined by a variety of methods includingbut not limited to, isoelectric focusing and various computer algorithms(see for example Bjellqvist et al., 1993, Electrophoresis 14:1023). Inone embodiment, the pI of the anti-EFNA antibodies of the invention isbetween is higher than about 6.5, about 7.0, about 7.5, about 8.0, about8.5, or about 9.0. In another embodiment, the pI of the anti-EFNAantibodies of the invention is between is higher than 6.5, 7.0, 7.5,8.0, 8.5, or 9.0. In yet another embodiment, substitutions resulting inalterations in the pI of antibodies of the invention will notsignificantly diminish their binding affinity for EFNA. As discussed inmore detail below, it is specifically contemplated that thesubstitution(s) of the Fc region that result in altered binding to FcγRmay also result in a change in the pI. In a preferred embodiment,substitution(s) of the Fc region are specifically chosen to effect boththe desired alteration in FcγR binding and any desired change in pI. Asused herein, the pI value is defined as the pI of the predominant chargeform.

g. Thermal Stability

It will further be appreciated that the Tm of the Fab domain of anantibody can be a good indicator of the thermal stability of an antibodyand may further provide an indication of the shelf-life. Tm is merelythe temperature of 50% unfolding for a given domain or sequence. A lowerTm indicates more aggregation/less stability, whereas a higher Tmindicates less aggregation/more stability. Thus, antibodies or fragmentsor derivatives having higher Tm are preferable. Moreover, usingart-recognized techniques it is possible to alter the composition of theanti-EFNA antibodies or domains thereof to increase or optimizemolecular stability. See, for example, U.S. Pat. No. 7,960,142. Thus, inone embodiment, the Fab domain of a selected antibody has a Tm valuehigher than at least 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80°C., 85° C., 90° C., 95° C., 100° C., 105° C., 110° C., 115° C. or 120°C. In another embodiment, the Fab domain of an antibody has a Tm valuehigher than at least about 50° C., about 55° C., about 60° C., about 65°C., about 70° C., about 75° C., about 80° C., about 85° C., about 90°C., about 95° C., about 100° C., about 105° C., about 110° C., about115° C. or about 120° C. Thermal melting temperatures (Tm) of a proteindomain (e.g., a Fab domain) can be measured using any standard methodknown in the art, for example, by differential scanning calorimetry(see, e.g., Vermeer et al., 2000, Biophys. J. 78:394-404; Vermeer etal., 2000, Biophys. J. 79: 2150-2154 both incorporated herein byreference).

VII. EFNA Modulator Fragments and Derivatives

Whether the agents of the present invention comprise intact fusionconstructs, antibodies, fragments or derivatives, the selectedmodulators will react, bind, combine, complex, connect, attach, join,interact or otherwise associate with EFNA and thereby provide thedesired anti-neoplastic effects. Those of skill in the art willappreciate that modulators comprising anti-EFNA antibodies interact orassociate with EFNA through one or more binding sites expressed on theantibody. More specifically, as used herein the term binding sitecomprises a region of a polypeptide that is responsible for selectivelybinding to a target molecule of interest (e.g., enzyme, antigen, ligand,receptor, substrate or inhibitor). Binding domains comprise at least onebinding site (e.g. an intact IgG antibody will have two binding domainsand two binding sites). Exemplary binding domains include an antibodyvariable domain, a receptor-binding domain of a ligand, a ligand-bindingdomain of a receptor or an enzymatic domain. For the purpose of theinstant invention the typical active region of EFNA (e.g., as part of anFc-EFNA fusion construct) may comprise a binding site for a substrate(e.g., an Eph receptor).

a. Fragments

Regardless of which form of the modulator (e.g. chimeric, humanized,etc.) is selected to practice the invention, it will be appreciated thatimmunoreactive fragments of the same may be used in accordance with theteachings herein. In the broadest sense, the term antibody fragmentcomprises at least a portion of an intact antibody (e.g. a naturallyoccurring immunoglobulin). More particularly the term fragment refers toa part or portion of an antibody or antibody chain (or EFNA molecule inthe case of Fc fusions) comprising fewer amino acid residues than anintact or complete antibody or antibody chain. The term antigen-bindingfragment refers to a polypeptide fragment of an immunoglobulin orantibody that binds antigen or competes with intact antibody (i.e., withthe intact antibody from which they were derived) for antigen binding(i.e., specific binding). As used herein, the term fragment of anantibody molecule includes antigen-binding fragments of antibodies, forexample, an antibody light chain (V_(L)), an antibody heavy chain(V_(H)), a single chain antibody (scFv), a F(ab′)2 fragment, a Fabfragment, an Fd fragment, an Fv fragment, single domain antibodyfragments, diabodies, linear antibodies, single-chain antibody moleculesand multispecific antibodies formed from antibody fragments. Similarly,an active fragment of EFNA comprises a portion of the EFNA molecule thatretains its ability to interact with EFNA substrates or receptors andmodify them in a manner similar to that of an intact EFNA (though maybewith somewhat less efficiency).

Those skilled in the art will appreciate fragments can be obtained viachemical or enzymatic treatment of an intact or complete modulator(e.g., antibody or antibody chain) or by recombinant means. In thisregard, while various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,explicitly includes antibodies or fragments or derivatives thereofeither produced by the modification of whole antibodies or synthesizedde novo using recombinant DNA methodologies.

More specifically, papain digestion of antibodies produces two identicalantigen-binding fragments, called Fab fragments, each with a singleantigen-binding site, and a residual Fc fragment, whose name reflectsits ability to crystallize readily. Pepsin treatment yields an F(ab′)₂fragment that has two antigen-binding sites and is still capable ofcross-linking antigen. The Fab fragment also contains the constantdomain of the light chain and the first constant domain (C_(H)1) of theheavy chain. Fab′ fragments differ from Fab fragments by the addition ofa few residues at the carboxy terminus of the heavy-chain C_(H)1 domainincluding one or more cysteines from the antibody hinge region. Fab′-SHis the designation herein for Fab′ in which the cysteine residue(s) ofthe constant domains bear at least one free thiol group. F(ab′)₂antibody fragments originally were produced as pairs of Fab′ fragmentsthat have hinge cysteines between them. Other chemical couplings ofantibody fragments are also known. See, e.g., Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1999), for a more detailed descriptionof other antibody fragments.

It will further be appreciated that an Fv fragment is an antibodyfragment that contains a complete antigen recognition and binding site.This region is made up of a dimer of one heavy and one light chainvariable domain in tight association, which can be covalent in nature,for example in scFv. It is in this configuration that the three CDRs ofeach variable domain interact to define an antigen binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs or a subsetthereof confer antigen binding specificity to the antibody. However,even a single variable domain (or half of an Fv comprising only threeCDRs specific for an antigen) has the ability to recognize and bindantigen, although usually at a lower affinity than the entire bindingsite.

In other embodiments an antibody fragment, for example, is one thatcomprises the Fc region, retains at least one of the biologicalfunctions normally associated with the Fc region when present in anintact antibody, such as FcRn binding, antibody half life modulation,ADCC function and complement binding. In one embodiment, an antibodyfragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For example, such anantibody fragment may comprise on antigen binding arm linked to an Fcsequence capable of conferring in vivo stability to the fragment.

b. Derivatives

In another embodiment, it will further be appreciated that themodulators of the invention may be monovalent or multivalent (e.g.,bivalent, trivalent, etc.). As used herein the term valency refers tothe number of potential target (i.e., EFNA) binding sites associatedwith an antibody. Each target binding site specifically binds one targetmolecule or specific position or locus on a target molecule. When anantibody of the instant invention comprises more than one target bindingsite (multivalent), each target binding site may specifically bind thesame or different molecules (e.g., may bind to different ligands ordifferent antigens, or different epitopes or positions on the sameantigen). For the purposes of the instant invention, the subjectantibodies will preferably have at least one binding site specific forhuman EFNA. In one embodiment the antibodies of the instant inventionwill be monovalent in that each binding site of the molecule willspecifically bind to a single EFNA position or epitope. In otherembodiments, the antibodies will be multivalent in that they comprisemore than one binding site and the different binding sites specificallyassociate with more than a single position or epitope. In such cases themultiple epitopes may be present on the selected EFNA polypeptide orspice variant or a single epitope may be present on EFNA while a second,different epitope may be present on another molecule or surface. See,for example, U.S.P.N. 2009/0130105.

As alluded to above, multivalent antibodies may immunospecifically bindto different epitopes of the desired target molecule or mayimmunospecifically bind to both the target molecule as well as aheterologous epitope, such as a heterologous polypeptide or solidsupport material. While preferred embodiments of the anti-EFNAantibodies only bind two antigens (i.e. bispecific antibodies),antibodies with additional specificities such as trispecific antibodiesare also encompassed by the instant invention. Examples of bispecificantibodies include, without limitation, those with one arm directedagainst EFNA and the other arm directed against any other antigen (e.g.,an modulator cell marker). Methods for making bispecific antibodies areknown in the art. Traditional production of full-length bispecificantibodies is based on the coexpression of two immunoglobulin heavychain-light chain pairs, where the two chains have differentspecificities (Millstein et al., 1983, Nature, 305:537-539). Other moresophisticated compatible multispecific constructs and methods of theirfabrication are set forth in U.S.P.N. 2009/0155255.

In yet other embodiments, antibody variable domains with the desiredbinding specificities (antibody-antigen combining sites) are fused toimmunoglobulin constant domain sequences. The fusion preferably is withan immunoglobulin heavy chain constant domain, comprising at least partof the hinge, C_(H)2, and/or C_(H)3 regions. In one example, the firstheavy-chain constant region (C_(H)1) containing the site necessary forlight chain binding is present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Thisprovides for great flexibility in adjusting the mutual proportions ofthe three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector when,the expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm (e.g., EFNA4), and a hybrid immunoglobulin heavychain-light chain pair (providing a second binding specificity) in theother arm. It was found that this asymmetric structure facilitates theseparation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation. This approach is disclosed in WO 94/04690. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., 1986, Methods in Enzymology, 121:210. According toanother approach described in WO96/27011, a pair of antibody moleculescan be engineered to maximize the percentage of heterodimers that arerecovered from recombinant cell culture. The preferred interfacecomprises at least a part of the C_(H)3 domain of an antibody constantdomain. In this method, one or more small amino acid side chains fromthe interface of the first antibody molecule are replaced with largerside chains (e.g. tyrosine or tryptophan). Compensatory cavities ofidentical or similar size to the large side chain(s) are created on theinterface of the second antibody molecule by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). This providesa mechanism for increasing the yield of the heterodimer over otherunwanted end-products such as homodimers.

Bispecific antibodies also include cross-linked or heteroconjugateantibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

VIII. EFNA Modulators—Constant Region Modifications

a. Fc Region and Fe Receptors

In addition to the various modifications, substitutions, additions ordeletions to the variable or binding region of the disclosed modulators(e.g., Fc-EFNA or anti-EFNA antibodies) set forth above, those skilledin the art will appreciate that selected embodiments of the presentinvention may also comprise substitutions or modifications of theconstant region (i.e. the Fc region). More particularly, it iscontemplated that the EFNA modulators of the invention may contain interalia one or more additional amino acid residue substitutions, mutationsand/or modifications which result in a compound with preferredcharacteristics including, but not limited to: altered pharmacokinetics,increased serum half life, increase binding affinity, reducedimmunogenicity, increased production, altered Fc ligand binding,enhanced or reduced ADCC or CDC activity, altered glycosylation and/ordisulfide bonds and modified binding specificity. In this regard it willbe appreciated that these Fe variants may advantageously be used toenhance the effective anti-neoplastic properties of the disclosedmodulators.

The term Fc region herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue. A functional Fe region possesses aneffector function of a native sequence Fc region. Exemplary effectorfunctions include C1q binding; CDC; Fc receptor binding; ADCC;phagocytosis; down regulation of cell surface receptors (e.g. B cellreceptor; BCR), etc. Such effector functions generally require the Fcregion to be combined with a binding domain (e.g., an antibody variabledomain) and can be assessed using various assays as disclosed, forexample, in definitions herein.

Fc receptor or FcR describes a receptor that binds to the Fc region ofan antibody. In some embodiments, an FcR is a native human FcR. In someembodiments, an FcR is one that binds an IgG antibody (a gamma receptor)and includes receptors of the FcγRI, Fc.RII, and FcγRIII subclasses,including allelic variants and alternatively spliced forms of thosereceptors. FcγII receptors include FcγRIIA (an activating receptor) andFcγRIIB (an inhibiting receptor), which have similar amino acidsequences that differ primarily in the cytoplasmic domains thereof.Activating receptor Fcγ RIIA contains an immunoreceptor tyrosine-basedactivation motif (ITAM) in its cytoplasmic domain. Inhibiting receptorFγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM)in its cytoplasmic domain. (see, e.g., Daeron, Annu. Rev. Immunol.15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch andKinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41(1995). Other FcRs, including those to be identified in the future, areencompassed by the term FcR herein. The term Fc receptor or FcR alsoincludes the neonatal receptor, FcRn, which, in certain instances, isresponsible for the transfer of maternal IgGs to the fetus (Guyer etal., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249(1994)) and regulation of homeostasis of immunoglobulins. Methods ofmeasuring binding to FcRn are known (see, e.g., Ghetie and Ward.,Immunol. Today 18(12):592-598 (1997); Ghetie et al., NatureBiotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem.279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).

b. Fc Functions

As used herein complement dependent cytotoxicity and CDC refer to thelysing of a target cell in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (C1q) to a molecule, an antibody for example,complexed with a cognate antigen. To assess complement activation, a CDCassay, e.g. as described in Gazzano-Santoro et al., 1996, J. Immunol.Methods, 202:163, may be performed.

Further, antibody-dependent cell-mediated cytotoxicity or ADCC refers toa form of cytotoxicity in which secreted Ig bound onto Fc receptors(FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK)cells, neutrophils, and macrophages) enables these cytotoxic effectorcells to bind specifically to an antigen-bearing target cell andsubsequently kill the target cell with cytotoxins. Specifichigh-affinity IgG antibodies directed to the target arm cytotoxic cellsand are absolutely required for such killing. Lysis of the target cellis extracellular, requires direct cell-to-cell contact, and does notinvolve complement.

EFNA modulator variants with altered FcR binding affinity or ADCCactivity is one which has either enhanced or diminished FcR bindingactivity and/or ADCC activity compared to a parent or unmodifiedantibody or to a modulator comprising a native sequence Fc region. Themodulator variant which displays increased binding to an FcR binds atleast one FcR with better affinity than the parent or unmodifiedantibody or to a modulator comprising a native sequence Fc region. Avariant which displays decreased binding to an FcR, binds at least oneFcR with worse affinity than the parent or unmodified antibody or to amodulator comprising a native sequence Fc region. Such variants whichdisplay decreased binding to an FcR may possess little or no appreciablebinding to an FcR, e.g., 0-20% binding to the FcR compared to a nativesequence IgG Fc region, e.g. as determined techniques well known in theart.

As to FcRn, the antibodies of the instant invention also comprise orencompass Fc variants with modifications to the constant region thatprovide half-lives (e.g., serum half-lives) in a mammal, preferably ahuman, of greater than 5 days, greater than 10 days, greater than 15days, preferably greater than 20 days, greater than 25 days, greaterthan 30 days, greater than 35 days, greater than 40 days, greater than45 days, greater than 2 months, greater than 3 months, greater than 4months, or greater than 5 months. The increased half-lives of theantibodies (or Fe containing molecules) of the present invention in amammal, preferably a human, results in a higher serum titer of saidantibodies or antibody fragments in the mammal, and thus, reduces thefrequency of the administration of said antibodies or antibody fragmentsand/or reduces the concentration of said antibodies or antibodyfragments to be administered. Antibodies having increased in vivohalf-lives can be generated by techniques known to those of skill in theart. For example, antibodies with increased in vivo half-lives can begenerated by modifying (e.g., substituting, deleting or adding) aminoacid residues identified as involved in the interaction between the Fcdomain and the FcRn receptor (see, e.g., International Publication Nos.WO 97/34631; WO 04/029207; U.S. Pat. No. 6,737,056 and U.S.P.N.2003/0190311. Binding to human FcRn in vivo and serum half life of humanFcRn high affinity binding polypeptides can be assayed, e.g., intransgenic mice or transfected human cell lines expressing human FcRn,or in primates to which the polypeptides with a variant Fe region areadministered. WO 2000/42072 describes antibody variants with improved ordiminished binding to FcRns. See also, e.g., Shields et al. J. Biol.Chem. 9(2):6591-6604 (2001).

c. Glycosylation Modifications

In still other embodiments, glycosylation patterns or compositions ofthe antibodies of the invention are modified. More particularly,preferred embodiments of the present invention may comprise one or moreengineered glycoforms, i.e., an altered glycosylation pattern or alteredcarbohydrate composition that is covalently attached to a moleculecomprising an Fc region. Engineered glycoforms may be useful for avariety of purposes, including but not limited to enhancing or reducingeffector function, increasing the affinity of the antibody for a targetantigen or facilitating production of the antibody. In cases wherereduced effector function is desired, it will be appreciated that themolecule may be engineered to express in an aglycosylated form. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Thatis, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site (see e.g. U.S. Pat. Nos.5,714,350 and 6,350,861. Conversely, enhanced effector functions orimproved binding may be imparted to the Fc containing molecule byengineering in one or more additional glycosylation sites.

Additionally or alternatively, an Fc variant can be made that has analtered glycosylation composition, such as a hypofucosylated antibodyhaving reduced amounts of fucosyl residues or an antibody havingincreased bisecting GlcNAc structures. These and similar alteredglycosylation patterns have been demonstrated to increase the ADCCability of antibodies. Engineered glycoforms may be generated by anymethod known to one skilled in the art, for example by using engineeredor variant expression strains, by coexpression with one or more enzymes(for example N-acetylglucosaminyltransferase III (GnTI11)), byexpressing a molecule comprising an Fc region in various organisms orcell lines from various organisms or by modifying carbohydrate(s) afterthe molecule comprising Fc region has been expressed. See, for example,Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana etal. (1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP1,176,195; PCT Publications WO 03/035835; WO 99/54342, Umana et al,1999, Nat. Biotechnol 17:176-180; Davies et al., 20017 Biotechnol Bioeng74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawaet al., 2003, J Biol Chem 278:3466-3473) U.S. Pat. No. 6,602,684; U.S.Ser. Nos. 10/277,370; 10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1;PCT WO 02/311140A1; PCT WO 02/30954A1; Potillegent™ technology (Biowa,Inc.); GlycoMAb™ glycosylation engineering technology (GLYCARTbiotechnology AG); WO 00061739; EA01229125; U.S.P.N. 2003/0115614;Okazaki et al., 2004, JMB, 336: 1239-49.

IX. Modulator Expression

a. Overview

DNA encoding the desired EFNA modulators may be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encodingantibody heavy and light chains). Isolated and subcloned hybridoma cells(or phage or yeast derived colonies) may serve as a preferred source ofsuch DNA if the modulator is an antibody. If desired, the nucleic acidcan further be manipulated as described herein to create agentsincluding fusion proteins, or chimeric, humanized or fully humanantibodies. More particularly, the isolated DNA (which may be modified)can be used to clone constant and variable region sequences for themanufacture antibodies as described in U.S. Pat. No. 7,709,611.

This exemplary method entails extraction of RNA from the selected cells,conversion to cDNA, and amplification by PCR using antibody specificprimers. Suitable primers are well known in the art and, as exemplifiedherein, are readily available from numerous commercial sources. It willbe appreciated that, to express a recombinant human or non-humanantibody isolated by screening of a combinatorial library, the DNAencoding the antibody is cloned into a recombinant expression vector andintroduced into host cells including mammalian cells, insect cells,plant cells, yeast, and bacteria. In yet other embodiments, themodulators are introduced into and expressed by simian COS cells, NS0cells, Chinese Hamster Ovary (CHO) cells or myeloma cells that do nototherwise produce the desired construct. As will be discussed in moredetail below, transformed cells expressing the desired modulator may begrown up in relatively large quantities to provide clinical andcommercial supplies of the fusion construct or immunoglobulin.

Whether the nucleic acid encoding the desired portion of the EFNAmodulator is obtained or derived from phage display technology, yeastlibraries, hybridoma based technology, synthetically or from commercialsources, it is to be understood that the present invention explicitlyencompasses nucleic acid molecules and sequences encoding EFNAmodulators including fusion proteins and anti-EFNA antibodies orantigen-binding fragments or derivatives thereof. The invention furtherencompasses nucleic acids or nucleic acid molecules (e.g.,polynucleotides) that hybridize under high stringency, or alternatively,under intermediate or lower stringency hybridization conditions (e.g.,as defined below), to polynucleotides complementary to nucleic acidshaving a polynucleotide sequence that encodes a modulator of theinvention or a fragment or variant thereof. The term nucleic acidmolecule or isolated nucleic acid molecule, as used herein, is intendedto include at least DNA molecules and RNA molecules. A nucleic acidmolecule may be single-stranded or double-stranded, but preferably isdouble-stranded DNA. Moreover, the present invention comprises anyvehicle or construct, incorporating such modulator encodingpolynucleotide including, without limitation, vectors, plasmids, hostcells, cosmids or viral constructs.

The term isolated nucleic acid means a that the nucleic acid was (i)amplified in vitro, for example by polymerase chain reaction (PCR), (ii)recombinantly produced by cloning, (iii) purified, for example bycleavage and gel-electrophoretic fractionation, or (iv) synthesized, forexample by chemical synthesis. An isolated nucleic acid is a nucleicacid that is available for manipulation by recombinant DNA techniques.

More specifically, nucleic acids that encode a modulator, including oneor both chains of an antibody of the invention, or a fragment,derivative, mutein, or variant thereof, polynucleotides sufficient foruse as hybridization probes, PCR primers or sequencing primers foridentifying, analyzing, mutating or amplifying a polynucleotide encodinga polypeptide, anti-sense nucleic acids for inhibiting expression of apolynucleotide, and complementary sequences of the foregoing are alsoprovided. The nucleic acids can be any length. They can be, for example,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250,300, 350, 400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or morenucleotides in length, and/or can comprise one or more additionalsequences, for example, regulatory sequences, and/or be part of a largernucleic acid, for example, a vector. These nucleic acids can besingle-stranded or double-stranded and can comprise RNA and/or DNAnucleotides, and artificial variants thereof (e.g., peptide nucleicacids). Nucleic acids encoding modulators of the invention, includingantibodies or immunoreactive fragments or derivatives thereof, havepreferably been isolated as described above.

b. Hybridization and Identity

As indicated, the invention further provides nucleic acids thathybridize to other nucleic acids under particular hybridizationconditions. Methods for hybridizing nucleic acids are well known in theart. See, e.g., Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989), 6.3.1-6.3.6. For the purposes of the instantapplication, a moderately stringent hybridization condition uses aprewashing solution containing 5× sodium chloride/sodium citrate (SSC),0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50%formamide, 6×SSC, and a hybridization temperature of 55° C. (or othersimilar hybridization solutions, such as one containing about 50%formamide, with a hybridization temperature of 42° C.), and washingconditions of 60° C., in 0.5×SSC, 0.1% SDS. A stringent hybridizationcondition hybridizes in 6×SSC at 45° C., followed by one or more washesin 0.1×SSC, 0.2% SDS at 68° C. Furthermore, one of skill in the art canmanipulate the hybridization and/or washing conditions to increase ordecrease the stringency of hybridization such that nucleic acidscomprising nucleotide sequences that are at least 65, 70, 75, 80, 85,90, 95, 98 or 99% identical to each other typically remain hybridized toeach other. More generally, for the purposes of the instant disclosurethe term substantially identical with regard to a nucleic acid sequencemay be construed as a sequence of nucleotides exhibiting at least about85%, or 90%, or 95%, or 97% sequence identity to the reference nucleicacid sequence.

The basic parameters affecting the choice of hybridization conditionsand guidance for devising suitable conditions are set forth by, forexample, Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., chapters 9 and 11; and Current Protocols in MolecularBiology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections2.10 and 6.3-6.4), and can be readily determined by those havingordinary skill in the art based on, for example, the length and/or basecomposition of the nucleic acid.

It will further be appreciated that nucleic acids may, according to theinvention, be present alone or in combination with other nucleic acids,which may be homologous or heterologous. In preferred embodiments, anucleic acid is functionally linked to expression control sequences thatmay be homologous or heterologous with respect to said nucleic acid. Inthis context the term homologous means that a nucleic acid is alsofunctionally linked to the expression control sequence naturally and theterm heterologous means that a nucleic acid is not functionally linkedto the expression control sequence naturally.

c. Expression

A nucleic acid, such as a nucleic acid expressing RNA and/or protein orpeptide, and an expression control sequence are functionally linked toone another, if they are covalently linked to one another in such a waythat expression or transcription of said nucleic acid is under thecontrol or under the influence of said expression control sequence. Ifthe nucleic acid is to be translated into a functional protein, then,with an expression control sequence functionally linked to a codingsequence, induction of said expression control sequence results intranscription of said nucleic acid, without causing a frame shift in thecoding sequence or said coding sequence not being capable of beingtranslated into the desired protein or peptide.

The term expression control sequence comprises according to theinvention promoters, ribosome binding sites, enhancers and other controlelements that regulate transcription of a gene or translation of mRNA.In particular embodiments of the invention, the expression controlsequences can be regulated. The exact structure of expression controlsequences may vary as a function of the species or cell type, butgenerally comprises 5′-untranscribed and 5′- and 3′-untranslatedsequences which are involved in initiation of transcription andtranslation, respectively, such as TATA box, capping sequence, CAATsequence, and the like. More specifically, 5′-untranscribed expressioncontrol sequences comprise a promoter region that includes a promotersequence for transcriptional control of the functionally linked nucleicacid. Expression control sequences may also comprise enhancer sequencesor upstream activator sequences.

According to the invention the term promoter or promoter region relatesto a nucleic acid sequence which is located upstream (5′) to the nucleicacid sequence being expressed and controls expression of the sequence byproviding a recognition and binding site for RNA-polymerase. Thepromoter region may include further recognition and binding sites forfurther factors that are involved in the regulation of transcription ofa gene. A promoter may control the transcription of a prokaryotic oreukaryotic gene. Furthermore, a promoter may be inducible and mayinitiate transcription in response to an inducing agent or may beconstitutive if transcription is not controlled by an inducing agent. Agene that is under the control of an inducible promoter is not expressedor only expressed to a small extent if an inducing agent is absent. Inthe presence of the inducing agent the gene is switched on or the levelof transcription is increased. This is mediated, in general, by bindingof a specific transcription factor.

Promoters which are preferred according to the invention includepromoters for SP6, T3 and T7 polymerase, human U6 RNA promoter, CMVpromoter, and artificial hybrid promoters thereof (e.g. CMV) where apart or parts are fused to a part or parts of promoters of genes ofother cellular proteins such as e.g. human GAPDH(glyceraldehyde-3-phosphate dehydrogenase), and including or notincluding (an) additional intron(s).

According to the invention, the term expression is used in its mostgeneral meaning and comprises the production of RNA or of RNA andprotein/peptide. It also comprises partial expression of nucleic acids.Furthermore, expression may be carried out transiently or stably.

In a preferred embodiment, a nucleic acid molecule is according to theinvention present in a vector, where appropriate with a promoter, whichcontrols expression of the nucleic acid. The term vector is used here inits most general meaning and comprises any intermediary vehicle for anucleic acid which enables said nucleic acid, for example, to beintroduced into prokaryotic and/or eukaryotic cells and, whereappropriate, to be integrated into a genome. Vectors of this kind arepreferably replicated and/or expressed in the cells. Vectors maycomprise plasmids, phagemids, bacteriophages or viral genomes. The termplasmid as used herein generally relates to a construct ofextrachromosomal genetic material, usually a circular DNA duplex, whichcan replicate independently of chromosomal DNA.

In practicing the present invention it will be appreciated that manyconventional techniques in molecular biology, microbiology, andrecombinant DNA technology are optionally used. Such conventionaltechniques relate to vectors, host cells and recombinant methods asdefined herein. These techniques are well known and are explained in,for example, Berger and Kimmel, Guide to Molecular Cloning Techniques,Methods in Enzymology volume 152 Academic Press, Inc., San Diego,Calif.; Sambrook et al., Molecular Cloning-A Laboratory Manual (3rdEd.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,2000 and Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., supra Other useful references, e.g. for cell isolation and culture(e.g., for subsequent nucleic acid or protein isolation) includeFreshney (1994) Culture of Animal Cells, a Manual of Basic Technique,third edition, Wiley-Liss, New York and the references cited therein;Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems JohnWiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips (Eds.) (1995)Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer LabManual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks(Eds.) The Handbook of Microbiological Media (1993) CRC Press, BocaRaton, Fla. Methods of making nucleic acids (e.g., by in vitroamplification, purification from cells, or chemical synthesis), methodsfor manipulating nucleic acids (e.g., site-directed mutagenesis, byrestriction enzyme digestion, ligation, etc.), and various vectors, celllines and the like useful in manipulating and making nucleic acids aredescribed in the above references. In addition, essentially anypolynucleotide (including, e.g., labeled or biotinylatedpolynucleotides) can be custom or standard ordered from any of a varietyof commercial sources.

Thus, in one aspect, the present invention provides recombinant hostcells allowing recombinant expression of antibodies of the invention orportions thereof. Antibodies produced by expression in such recombinanthost cells are referred to herein as recombinant antibodies. The presentinvention also provides progeny cells of such host cells, and antibodiesproduced by the same.

The term recombinant host cell (or simply host cell), as used herein,means a cell into which a recombinant expression vector has beenintroduced. It should be understood that recombinant host cell and hostcell mean not only the particular subject cell but also the progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term host cell as used herein. Suchcells may comprise a vector according to the invention as describedabove.

In another aspect, the present invention provides a method for making anantibody or portion thereof as described herein. According to oneembodiment, said method comprises culturing a cell transfected ortransformed with a vector as described above, and retrieving theantibody or portion thereof.

As indicated above, expression of an antibody of the invention (orfragment or variants thereof) preferably comprises expression vector(s)containing a polynucleotide that encodes the desired anti-EFNA antibody.Methods that are well known to those skilled in the art can be used toconstruct expression vectors comprising antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Embodiments ofthe invention, thus, provide replicable vectors comprising a nucleotidesequence encoding an anti-EFNA antibody of the invention (e.g., a wholeantibody, a heavy or light chain of an antibody, a heavy or light chainvariable domain of an antibody, or a portion thereof, or a heavy orlight chain CDR, a single chain Fv, or fragments or variants thereof),operably linked to a promoter. In preferred embodiments such vectors mayinclude a nucleotide sequence encoding the heavy chain of an antibodymolecule (or fragment thereof), a nucleotide sequence encoding the lightchain of an antibody (or fragment thereof) or both the heavy and lightchain.

Once the nucleotides of the present invention have been isolated andmodified according to the teachings herein, they may be used to produceselected modulators including anti-EFNA antibodies or fragments thereof.

X. Modulator Production and Purification

Using art recognized molecular biology techniques and current proteinexpression methodology, substantial quantities of the desired modulatorsmay be produced. More specifically, nucleic acid molecules encodingmodulators, such as antibodies obtained and engineered as describedabove, may be integrated into well known and commercially availableprotein production systems comprising various types of host cells toprovide preclinical, clinical or commercial quantities of the desiredpharmaceutical product. It will be appreciated that in preferredembodiments the nucleic acid molecules encoding the modulators areengineered into vectors or expression vectors that provide for efficientintegration into the selected host cell and subsequent high expressionlevels of the desired EFNA modulator.

Preferably nucleic acid molecules encoding EFNA modulators and vectorscomprising these nucleic acid molecules can be used for transfection ofa suitable mammalian, plant, bacterial or yeast host cell though it willbe appreciated that prokaryotic systems may be used for modulatorproduction. Transfection can be by any known method for introducingpolynucleotides into a host cell. Methods for the introduction ofheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei. In addition, nucleicacid molecules may be introduced into mammalian cells by viral vectors.Methods of transforming mammalian cells are well known in the art. See,e.g., U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455.Further, methods of transforming plant cells are well known in the art,including, e.g., Agrobacterium-mediated transformation, biolistictransformation, direct injection, electroporation and viraltransformation. Methods of transforming bacterial and yeast cells arealso well known in the art.

Moreover, the host cell may be co-transfected with two expressionvectors of the invention, for example, the first vector encoding a heavychain derived polypeptide and the second vector encoding a light chainderived polypeptide. The two vectors may contain identical selectablemarkers that enable substantially equal expression of heavy and lightchain polypeptides. Alternatively, a single vector may be used whichencodes, and is capable of expressing, both heavy and light chainpolypeptides. In such situations, the light chain is preferably placedbefore the heavy chain to avoid an excess of toxic free heavy chain. Thecoding sequences for the heavy and light chains may comprise cDNA orgenomic DNA.

a. Host-Expression Systems

A variety of host-expression vector systems, many commerciallyavailable, are compatible with the teachings herein and may be used toexpress the modulators of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beexpressed and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express a molecule of the invention in situ. Such systemsinclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli, B. subtilis, streptomyces) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining modulator coding sequences; yeast (e.g., Saccharomyces,Pichia) transfected with recombinant yeast expression vectors containingmodulator coding sequences; insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus) containingmodulator coding sequences; plant cell systems (e.g., Nicotiana,Arabidopsis, duckweed, corn, wheat, potato, etc.) infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transfected with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing modulator codingsequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the moleculebeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of pharmaceutical compositions of amodulator, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited to, the E. coli expression vectorpUR278 (Ruther et al., EMBO 1. 2:1791 (1983)), in which the codingsequence may be ligated individually into the vector in frame with thelac Z coding region so that a fusion protein is produced; pIN vectors(Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke &Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEXvectors may also be used to express foreign polypeptides as fusionproteins with glutathione 5-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption and binding to matrix glutathione agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) may be used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The coding sequences may be clonedindividually into non-essential regions (for example, the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample, the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe used to introduce the desired nucleotide sequence. In cases where anadenovirus is used as an expression vector, the coding sequence ofinterest may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing the molecule in infectedhosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 8 1:355-359(1984)). Specific initiation signals may also be required for efficienttranslation of inserted coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see, e.g., Bittner et al.,Methods in Enzymol. 153:51-544 (1987)). Thus, compatible mammalian celllines available as hosts for expression are well known in the art andinclude many immortalized cell lines available from the American TypeCulture Collection (ATCC). These include, inter alia, Chinese hamsterovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 Freestylecells (Life Technologies), NIH-3T3 cells, HeLa cells, baby hamsterkidney (BHK) cells, African green monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a numberof other cell lines.

For long-term, high-yield production of recombinant proteins stableexpression is preferred. Accordingly, cell lines that stably express theselected modulator may be engineered using standard art recognizedtechniques. Rather than using expression vectors that contain viralorigins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA, engineered cells may be allowed to grow for 1-2 days in anenriched media, and then are switched to a selective media. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci which in turn can be cloned andexpanded into cell lines. This method may advantageously be used toengineer cell lines which express the molecule. Such engineered celllines may be particularly useful in screening and evaluation ofcompositions that interact directly or indirectly with the molecule.

A number of selection systems are well known in the art and may be usedincluding, but not limited to, the herpes simplex virus thymidine kinase(Wigler et al., Cell 11:223 (1977)), hypoxanthineguaninephosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci.USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al.,Cell 22:8 17 (1980)) genes can be employed in tk-, hgprt- or aprt-cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for the following genes: dhfr, which confers resistance tomethotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hareet al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confersresistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci.USA 78:2072 (1981)); neo, which confers resistance to the aminoglycosideG-418 (Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62: 191-217 (1993); TIB TECH 11(5):155-2 15 (May, 1993)); andhygro, which confers resistance to hygromycin (Santerre et al., Gene30:147 (1984)). Methods commonly known in the art of recombinant DNAtechnology may be routinely applied to select the desired recombinantclone, and such methods are described, for example, in Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY(1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al.(eds), Current Protocols in Human Genetics, John Wiley & Sons, NY(1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981). It will beappreciated that one particularly preferred method of establishing astable, high yield cell line comprises the glutamine synthetase geneexpression system (the GS system) which provides an efficient approachfor enhancing expression under certain conditions. The GS system isdiscussed in whole or part in connection with EP patents 0 216 846, 0256 055, 0 323 997 and 0 338 841 each of which is incorporated herein byreference.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function and/or purification of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. As known in the art appropriate cell lines or host systems canbe chosen to ensure the desired modification and processing of theexpressed polypeptide. To this end, eukaryotic host cells that possessthe cellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product are particularlyeffective for use in the instant invention. Accordingly, particularlypreferred mammalian host cells include, but are not limited to, CHO,VERY, BHK, HeLa, COS, NS0, MDCK, 293, 3T3, W138, as well as breastcancer cell lines such as, for example, BT483, Hs578T, HTB2, BT2O andT47D, and normal mammary gland cell line such as, for example, CRL7O3Oand HsS78Bst. Depending on the modulator and the selected productionsystem, those of skill in the art may easily select and optimizeappropriate host cells for efficient expression of the modulator.

b. Chemical Synthesis

Besides the aforementioned host cell systems, it will be appreciatedthat the modulators of the invention may be chemically synthesized usingtechniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., N.Y., andHunkapiller, M., et al., 1984, Nature 310:105-111). For example, apeptide corresponding to a polypeptide fragment of the invention can besynthesized by use of a peptide synthesizer. Furthermore, if desired,nonclassical amino acids or chemical amino acid analogs can beintroduced as a substitution or addition into a polypeptide sequence.Non-classical amino acids include, but are not limited to, to theD-isomers of the common amino acids, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu,e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine,fluoro-amino acids, designer amino acids such as b-methyl amino acids,Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs ingeneral. Furthermore, the amino acid can be D (dextrorotary) or L(levorotary).

c. Transgenic Systems

The EFNA modulators of the invention also can be produced transgenicallythrough the generation of a mammal or plant that is transgenic for theimmunoglobulin heavy and light chain sequences (or fragments orderivatives or variants thereof) of interest and production of thedesired compounds in a recoverable form. In connection with thetransgenic production in mammals, anti-EFNA antibodies, for example, canbe produced in, and recovered from, the milk of goats, cows, or othermammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and5,741,957. In some embodiments, non-human transgenic animals thatcomprise human immunoglobulin loci are immunized with EFNA or animmunogenic portion thereof, as described above. Methods for makingantibodies in plants are described, e.g., in U.S. Pat. Nos. 6,046,037and 5,959,177.

In accordance with the teachings herein non-human transgenic animals orplants may be produced by introducing one or more nucleic acid moleculesencoding an EFNA modulator of the invention into the animal or plant bystandard transgenic techniques. See Hogan and U.S. Pat. No. 6,417,429.The transgenic cells used for making the transgenic animal can beembryonic stem cells or somatic cells or a fertilized egg. Thetransgenic non-human organisms can be chimeric, nonchimericheterozygotes, and nonchimeric homozygotes. See, e.g., Hogan et al.,Manipulating the Mouse Embryo: A Laboratory Manual 2nd ed., Cold SpringHarbor Press (1999); Jackson et al., Mouse Genetics and Transgenics: APractical Approach, Oxford University Press (2000); and Pinkert,Transgenic Animal Technology: A Laboratory Handbook, Academic Press(1999). In some embodiments, the transgenic non-human animals have atargeted disruption and replacement by a targeting construct thatencodes, for example, a heavy chain and/or a light chain of interest. Inone embodiment, the transgenic animals comprise and express nucleic acidmolecules encoding heavy and light chains that specifically bind toEFNA. While anti-EFNA antibodies may be made in any transgenic animal,in particularly preferred embodiments the non-human animals are mice,rats, sheep, pigs, goats, cattle or horses. In further embodiments thenon-human transgenic animal expresses the desired pharmaceutical productin blood, milk, urine, saliva, tears, mucus and other bodily fluids fromwhich it is readily obtainable using art recognized purificationtechniques.

It is likely that modulators, including antibodies, expressed bydifferent cell lines or in transgenic animals will have differentglycosylation patterns from each other. However, all modulators encodedby the nucleic acid molecules provided herein, or comprising the aminoacid sequences provided herein are part of the instant invention,regardless of the glycosylation state of the molecule, and moregenerally, regardless of the presence or absence of post-translationalmodification(s). In addition the invention encompasses modulators thatare differentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques, includingbut not limited, to specific chemical cleavage by cyanogen bromide,trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation,formylation, oxidation, reduction, metabolic synthesis in the presenceof tunicamycin, etc. Various post-translational modifications are alsoencompassed by the invention include, for example, e.g., N-linked orO-linked carbohydrate chains, processing of N-terminal or C-terminalends), attachment of chemical moieties to the amino acid backbone,chemical modifications of N-linked or O-linked carbohydrate chains, andaddition or deletion of an N-terminal methionine residue as a result ofprokaryotic host cell expression. Moreover, as set forth in the text andExamples below the polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, radioisotopic or affinitylabel to allow for detection and isolation of the modulator.

d. Purification

Once a modulator of the invention has been produced by recombinantexpression or any one of the other techniques disclosed herein, it maybe purified by any method known in the art for purification ofimmunoglobulins, or more generally by any other standard technique forthe purification of proteins. In this respect the modulator may beisolated. As used herein, an isolated EFNA modulator is one that hasbeen identified and separated and/or recovered from a component of itsnatural environment. Contaminant components of its natural environmentare materials that would interfere with diagnostic or therapeutic usesfor the polypeptide and may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. Isolated modulators include amodulator in situ within recombinant cells because at least onecomponent of the polypeptide's natural environment will not be present.

When using recombinant techniques, the EFNA modulator (e.g. an anti-EFNAantibody or derivative or fragment thereof) can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the desired molecule is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed fragments, maybe removed, for example, by centrifugation or ultrafiltration. Forexample, Carter, et al., Bio/Technology 10:163 (1992) describe aprocedure for isolating antibodies that are secreted to the periplasmicspace of E. coli. Briefly, cell paste is thawed in the presence ofsodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF)over about 30 minutes. Cell debris can be removed by centrifugation.Where the antibody is secreted into the medium, supernatants from suchexpression systems are generally first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. A protease inhibitor such asPMSF may be included in any of the foregoing steps to inhibitproteolysis and antibiotics may be included to prevent the growth ofadventitious contaminants.

The modulator (e.g., fc-EFNA or anti-EFNA antibody) composition preparedfrom the cells can be purified using, for example, hydroxylapatitechromatography, gel electrophoresis, dialysis, and affinitychromatography, with affinity chromatography being the preferredpurification technique. The suitability of protein A as an affinityligand depends on the species and isotype of any immunoglobulin Fcdomain that is present in the selected construct. Protein A can be usedto purify antibodies that are based on human IgG1, IgG2 or IgG4 heavychains (Lindmark, et al., J Immunol Meth 62:1 (1983)). Protein G isrecommended for all mouse isotypes and for human IgG3 (Guss, et al.,EMBO J 5:1567 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker;Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, reverse phase HPLC, chromatography on silica,chromatography on heparin, sepharose chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered. In particularlypreferred embodiments the modulators of the instant invention will bepurified, at least in part, using Protein A or Protein G affinitychromatography.

XI. Conjugated EFNA Modulators

Once the modulators of the invention have been purified according to theteachings herein they may be linked with, fused to, conjugated to (e.g.,covalently or non-covalently) or otherwise associated withpharmaceutically active or diagnostic moieties or biocompatiblemodifiers. As used herein the term conjugate will be used broadly andheld to mean any molecule associated with the disclosed modulatorsregardless of the method of association. In this respect it will beunderstood that such conjugates may comprise peptides, polypeptides,proteins, polymers, nucleic acid molecules, small molecules, mimeticagents, synthetic drugs, inorganic molecules, organic molecules andradioisotopes. Moreover, as indicated above the selected conjugate maybe covalently or non-covalently linked to the modulator and exhibitvarious molar ratios depending, at least in part, on the method used toeffect the conjugation.

In preferred embodiments it will be apparent that the modulators of theinvention may be conjugated or associated with proteins, polypeptides orpeptides that impart selected characteristics (e.g., biotoxins,biomarkers, purification tags, etc.). More generally, in selectedembodiments the present invention encompasses the use of modulators orfragments thereof recombinantly fused or chemically conjugated(including both covalent and non-covalent conjugations) to aheterologous protein or polypeptide wherein the polypeptide comprises atleast 10, at least 20, at least 30, at least 40, at least 50, at least60, at least 70, at least 80, at least 90 or at least 100 amino acids.The construct does not necessarily need to be directly linked, but mayoccur through linker sequences. For example, antibodies may be used totarget heterologous polypeptides to particular cell types expressingEFNA, either in vitro or in vivo, by fusing or conjugating themodulators of the present invention to antibodies specific forparticular cell surface receptors. Moreover, modulators fused orconjugated to heterologous polypeptides may also be used in in vitroimmunoassays and may be compatible with purification methodology knownin the art. See e.g., International publication No. WO 93/21232;European Patent No. EP 439,095; Naramura et al., 1994, Immunol. Lett.39:91-99; U.S. Pat. No. 5,474,981; Gillies et al., 1992, PNAS89:1428-1432; and Fell et al., 1991, J. Immunol. 146:2446-2452.

a. Biocompatible Modifiers

In a preferred embodiment, the modulators of the invention may beconjugated or otherwise associated with biocompatible modifiers that maybe used to adjust, alter, improve or moderate modulator characteristicsas desired. For example, antibodies or fusion constructs with increasedin vivo half-lives can be generated by attaching relatively highmolecular weight polymer molecules such as commercially availablepolyethylene glycol (PEG) or similar biocompatible polymers. Thoseskilled in the art will appreciate that PEG may be obtained in manydifferent molecular weight and molecular configurations that can beselected to impart specific properties to the antibody (e.g. thehalf-life may be tailored). PEG can be attached to modulators orantibody fragments or derivatives with or without a multifunctionallinker either through site-specific conjugation of the PEG to the N- orC-terminus of said antibodies or antibody fragments or via epsilon-aminogroups present on lysine residues. Linear or branched polymerderivatization that results in minimal loss of biological activity maybe used. The degree of conjugation can be closely monitored by SDS-PAGEand mass spectrometry to ensure optimal conjugation of PEG molecules toantibody molecules. Unreacted PEG can be separated from antibody-PEGconjugates by, e.g., size exclusion or ion-exchange chromatography. In asimilar manner, the disclosed modulators can be conjugated to albumin inorder to make the antibody or antibody fragment more stable in vivo orhave a longer half life in vivo. The techniques are well known in theart, see e.g., International Publication Nos. WO 93/15199, WO 93/15200,and WO 01/77137; and European Patent No. 0 413, 622. Other biocompatibleconjugates are evident to those of ordinary skill and may readily beidentified in accordance with the teachings herein.

b. Diagnostic or Detection Agents

In other preferred embodiments, modulators of the present invention, orfragments or derivatives thereof, are conjugated to a diagnostic ordetectable agent, marker or reporter which may be a biological molecule(e.g., a peptide or nucleotide), a small molecule, flourophore, orradioisotope. Labeled modulators can be useful for monitoring thedevelopment or progression of a hyperproliferative disorder or as partof a clinical testing procedure to determine the efficacy of aparticular therapy including the disclosed modulators (i.e.theragnostics). Such markers or reporters may also be useful inpurifying the selected modulator, separating or isolating TIC or inpreclinical procedures or toxicology studies.

Such diagnosis and detection can be accomplished by coupling themodulator to detectable substances including, but not limited to,various enzymes comprising for example horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; prostheticgroups, such as but not limited to streptavidinlbiotin andavidin/biotin; fluorescent materials, such as but not limited to,umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine,dichlorotriazinyl amine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as but not limited to, luminol;bioluminescent materials, such as but not limited to, luciferase,luciferin, and aequorin; radioactive materials, such as but not limitedto iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium(³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm,¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and¹¹⁷Tin; positron emitting metals using various positron emissiontomographies, noradioactive paramagnetic metal ions, and molecules thatare radiolabeled or conjugated to specific radioisotopes. In suchembodiments appropriate detection methodology is well known in the artand readily available from numerous commercial sources.

As indicated above, in other embodiments the modulators or fragmentsthereof can be fused to marker sequences, such as a peptide orfluorophore to facilitate purification or diagnostic procedures such asimmunohistochemistry or FACs. In preferred embodiments, the marker aminoacid sequence is a hexa-histidine (SEQ ID NO: 166) peptide, such as thetag provided in a pQE vector (Qiagen), among others, many of which arecommercially available. As described in Gentz et al., 1989, Proc. Natl.Acad. Sci. USA 86:821-824, for instance, hexa-histidine (SEQ ID NO: 166)provides for convenient purification of the fusion protein. Otherpeptide tags useful for purification include, but are not limited to,the hemagglutinin “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767)and the “flag” tag (U.S. Pat. No. 4,703,004).

c. Therapeutic Moieties

As previously alluded to the modulators or fragments or derivativesthereof may also be conjugated, linked or fused to or otherwiseassociated with a therapeutic moiety such as anti-cancer agents, acytotoxin or cytotoxic agent, e.g., a cytostatic or cytocidal agent, atherapeutic agent or a radioactive metal ion, e.g., alpha orbeta-emitters. As used herein a cytotoxin or cytotoxic agent includesany agent or therapeutic moiety that is detrimental to cells and mayinhibit cell growth or survival. Examples include paclitaxel,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4(Immunogen, Inc.), dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, puromycin, epirubicin, and cyclophosphamide and analogs orhomologs thereof. Additional cytoxins comprise auristatins, includingmonomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF)(Seattle Genetics, Inc.), amanitins such as alpha-amanitin,beta-amanitin, gamma-amanitin or epsilon-amanitin (Heidelberg PharmaAG), DNA minor groove binding agents such as duocarmycin derivatives(Syntarga, B.V.) and modified pyrrolobenzodiazepine dimers (PBDs,Spirogen, Ltd). Furthermore, in one embodiment the EFNA modulators ofthe instant invention may be associated with anti-CD3 binding moleculesto recruit cytotoxic T-cells and have them target the tumor initiatingcells (BiTE technology; see e.g., Fuhrmann, S. et. al. Annual Meeting ofAACR Abstract No. 5625 (2010) which is incorporated herein byreference).

Additional compatible therapeutic moieties comprise cytotoxic agentsincluding, but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), and anti-mitotic agents (e.g., vincristine andvinblastine). A more extensive list of therapeutic moieties can be foundin PCT publication WO 03/075957 and U.S.P.N. 2009/0155255 each of whichis incorporated herein by reference.

The selected modulators can also be conjugated to therapeutic moietiessuch as radioactive materials or macrocyclic chelators useful forconjugating radiometal ions (see above for examples of radioactivematerials). In certain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4:2483; Peterson et al., 1999, Bioconjug. Chem.10:553; and Zimmerman a al., 1999, Nucl. Med. Biol. 26:943.

Exemplary radioisotopes that may be compatible with this aspect of theinvention include, but are not limited to, iodine iodine (¹³¹I, ¹²⁵I,¹²³I, ¹²¹I), carbon (¹⁴C), copper (⁶²Cu, ⁶⁴Cu, ⁶⁷Cu), sulfur (³⁵S),tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In), bismuth (²¹²Bi,²¹³Bi), technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga),palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F),¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb,⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Tin, ²²⁵Ac, ⁷⁶Br, and ²¹¹At. Otherradionuclides are also available as diagnostic and therapeutic agents,especially those in the energy range of 60 to 4,000 keV. Depending onthe condition to be treated and the desired therapeutic profile, thoseskilled in the art may readily select the appropriate radioisotope foruse with the disclosed modulators.

EFNA modulators of the present invention may also be conjugated to atherapeutic moiety or drug that modifies a given biological response(e.g., biological response modifiers or BRMs). That is, therapeuticagents or moieties compatible with the instant invention are not to beconstrued as limited to classical chemical therapeutic agents. Forexample, in particularly preferred embodiments the drug moiety may be aprotein or polypeptide or fragment thereof possessing a desiredbiological activity. Such proteins may include, for example, a toxinsuch as abrin, ricin A, Onconase (or another cytotoxic RNase),pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein suchas tumor necrosis factor, α-interferon, β-interferon, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator, anapoptotic agent, e.g., TNF-α, TNF-β, AIM I (see, InternationalPublication No. WO 97/33899), AIM II (see, International Publication No.WO 97/34911), Fas Ligand (Takahashi et al., 1994, J. Immunol., 6:1567),and VEGI (see, International Publication No. WO 99/23105), a thromboticagent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or,a biological response modifier such as, for example, a lymphokine (e.g.,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), andgranulocyte colony stimulating factor (“G-CSF”)), or a growth factor(e.g., growth hormone (“GH”)). As set forth above, methods for fusing orconjugating modulators to polypeptide moieties are known in the art. Inaddition to the previously disclosed subject references see, e.g., U.S.Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851, and5,112,946; EP 307,434; EP 367,166; PCT Publications WO 96/04388 and WO91/06570; Ashkenazi et al., 1991, PNAS USA 88:10535; Zheng et al., 1995,J Immunol 154:5590; and Vil et al., 1992, PNAS USA 89:11337 each ofwhich is incorporated herein by reference. The association of amodulator with a moiety does not necessarily need to be direct, but mayoccur through linker sequences. Such linker molecules are commonly knownin the art and described in Denardo et al., 1998, Clin Cancer Res4:2483; Peterson et al., 1999, Bioconjug Chem 10:553; Zimmerman et al.,1999, Nucl Med Biol 26:943; Garnett, 2002, Adv Drug Deliv Rev 53:171each of which is incorporated herein.

More generally, techniques for conjugating therapeutic moieties orcytotoxic agents to modulators are well known. Moieties can beconjugated to modulators by any art-recognized method, including, butnot limited to aldehyde/Schiff linkage, sulphydryl linkage, acid-labilelinkage, cis-aconityl linkage, hydrazone linkage, enzymaticallydegradable linkage (see generally Garnett, 2002, Adv Drug Deliv Rev53:171). Also see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119. In preferred embodiments an EFNA modulator that isconjugated to a therapeutic moiety or cytotoxic agent may beinternalized by a cell upon binding to an EFNA molecule associated withthe cell surface thereby delivering the therapeutic payload.

XII. Diagnostics and Screening

a. Diagnostics

As indicated, the present invention provides in vitro or in vivo methodsfor detecting, diagnosing or monitoring hyperproliferative disorders andmethods of screening cells from a patient to identify tumorigenic cellsincluding TPCs. Such methods include identifying an individual havingcancer for treatment or monitoring progression of a cancer comprisingcontacting the patient or a sample obtained from a patient with aselected EFNA modulator as described herein and detecting presence orabsence, or level of association of the modulator to bound or freeephrin-A ligand in the sample. When the modulator comprises an antibodyor immunologically active fragment thereof the association withparticular EFNA in the sample likely denotes that the sample may containtumor perpetuating cells (e.g., a cancer stem cells) indicating that theindividual having cancer may be effectively treated with an EFNAmodulator as described herein. The methods may further comprise a stepof comparing the level of binding to a control. Conversely, when theselected modulator is Fc-EFNA the binding properties of the selectedephrin-A ligand may be exploited and monitored (directly or indirectly,in vivo or in vitro) when in contact with the sample to provide thedesired information. Other diagnostic or theragnostic methods compatiblewith the teachings herein are well known in the art and can be practicedusing commercial materials such as dedicated reporting systems.

In a particularly preferred embodiment the modulators of the instantinvention may be used to detect and quantify EFNA levels in a patientsample (e.g., plasma or blood) which may, in turn, be used to detect,diagnose or monitor EFNA associated disorders includinghyperproliferative disorders. One such embodiment is set forth inExample 17 below which provides for the detection of EFNA in plasmasamples.

Exemplary compatible assay methods include radioimmunoassays, enzymeimmunoassays, competitive-binding assays, fluorescent immunoassay,immunoblot assays, Western Blot analysis, flow cytometry assays, andELISA assays. More generally detection of EFNA in a biological sample orthe measurement of EFNA enzymatic activity (or inhibition thereof) maybe accomplished using any art-known assay. Compatible in vivotheragnostics or diagnostics may comprise art recognized imaging ormonitoring techniques such as magnetic resonance imaging (MRI),computerized tomography (e.g. CAT scan), positron tomography (e.g., PETscan) radiography, ultrasound, etc. Those skilled in the art willreadily be able to recognize and implement appropriate detection,monitoring or imaging techniques (often comprising commerciallyavailable sources) based on the etiology, pathological manifestation orclinical progression of the disorder.

In another embodiment, the invention provides a method of analyzingcancer progression and/or pathogenesis in-vivo. In another embodiment,analysis of cancer progression and/or pathogenesis in-vivo comprisesdetermining the extent of tumor progression. In another embodiment,analysis comprises the identification of the tumor. In anotherembodiment, analysis of tumor progression is performed on the primarytumor. In another embodiment, analysis is performed over time dependingon the type of cancer as known to one skilled in the art. In anotherembodiment, further analysis of secondary tumors originating frommetastasizing cells of the primary tumor is analyzed in-vivo. In anotherembodiment, the size and shape of secondary tumors are analyzed. In someembodiments, further ex-vivo analysis is performed.

In another embodiment, the invention provides a method of analyzingcancer progression and/or pathogenesis in-vivo including determiningcell metastasis. In yet another embodiment, analysis of cell metastasiscomprises determination of progressive growth of cells at a site that isdiscontinuous from the primary tumor. In another embodiment, the site ofcell metastasis analysis comprises the route of neoplastic spread. Insome embodiment, cells can disperse via blood vasculature, lymphatics,within body cavities or combinations thereof. In another embodiment,cell metastasis analysis is performed in view of cell migration,dissemination, extravasation, proliferation or combinations thereof.

In certain examples, the tumorigenic cells in a subject or a sample froma subject may be assessed or characterized using the disclosedmodulators prior to therapy or regimen to establish a baseline. In otherexamples the sample is derived from a subject that was treated. In someexamples the sample is taken from the subject at least about 1, 2, 4, 6,7, 8, 10, 12, 14, 15, 16, 18, 20, 30, 60, 90 days, 6 months, 9 months,12 months, or >12 months after the subject begins or terminatestreatment. In certain examples, the tumorigenic cells are assessed orcharacterized after a certain number of doses (e.g., after 2, 5, 10, 20,30 or more doses of a therapy). In other examples, the tumorigenic cellsare characterized or assessed after 1 week, 2 weeks, 1 month, 2 months,1 year, 2 years, 3 years, 4 years or more after receiving one or moretherapies.

In another aspect, and as discussed in more detail below, the presentinvention provides kits for detecting, monitoring or diagnosing ahyperproliferative disorder, identifying individual having such adisorder for possible treatment or monitoring progression (orregression) of the disorder in a patient, wherein the kit comprises amodulator as described herein, and reagents for detecting the impact ofthe modulator on a sample.

b. Screening

The EFNA modulators and cells, cultures, populations and compositionscomprising the same, including progeny thereof, can also be used toscreen for or identify compounds or agents (e.g., drugs) that affect afunction or activity of tumor initiating cells or progeny thereof byinteracting with an ephrin-A ligand (e.g., the polypeptide or geneticcomponents thereof). The invention therefore further provides systemsand methods for evaluation or identification of a compound or agent thatcan affect a function or activity tumor initiating cells or progenythereof by associating with EFNA or its substrates. Such compounds andagents can be drug candidates that are screened for the treatment of ahyperproliferative disorder, for example. In one embodiment, a system ormethod includes tumor initiating cells exhibiting EFNA and a compound oragent (e.g., drug), wherein the cells and compound or agent (e.g., drug)are in contact with each other.

The invention further provides methods of screening and identifying EFNAmodulators or agents and compounds for altering an activity or functionof tumor initiating cells or progeny cells. In one embodiment, a methodincludes contacting tumor initiating cells or progeny thereof with atest agent or compound; and determining if the test agent or compoundmodulates an activity or function of the ephrin-A ligand associatedtumor initiating cells.

A test agent or compound modulating an EFNA related activity or functionof such tumor initiating cells or progeny thereof within the populationidentifies the test agent or compound as an active agent. Exemplaryactivity or function that can be modulated include changes in cellmorphology, expression of a marker, differentiation ordedifferentiation, maturation, proliferation, viability, apoptosis orcell death neuronal progenitor cells or progeny thereof.

Contacting, when used in reference to cells or a cell culture or methodstep or treatment, means a direct or indirect interaction between thecomposition (e.g., an ephrin-A ligand associated cell or cell culture)and another referenced entity. A particular example of a directinteraction is physical interaction. A particular example of an indirectinteraction is where a composition acts upon an intermediary moleculewhich in turn acts upon the referenced entity (e.g., cell or cellculture).

In this aspect of the invention modulates indicates influencing anactivity or function of tumor initiating cells or progeny cells in amanner compatible with detecting the effects on cell activity orfunction that has been determined to be relevant to a particular aspect(e.g., metastasis or proliferation) of the tumor initiating cells orprogeny cells of the invention. Exemplary activities and functionsinclude, but are not limited to, measuring morphology, developmentalmarkers, differentiation, proliferation, viability, cell respiration,mitochondrial activity, membrane integrity, or expression of markersassociated with certain conditions. Accordingly, a compound or agent(e.g., a drug candidate) can be evaluated for its effect on tumorinitiating cells or progeny cells, by contacting such cells or progenycells with the compound or agent and measuring any modulation of anactivity or function of tumor initiating cells or progeny cells asdisclosed herein or would be known to the skilled artisan.

Methods of screening and identifying agents and compounds include thosesuitable for high throughput screening, which include arrays of cells(e.g., microarrays) positioned or placed, optionally at pre-determinedlocations or addresses. High-throughput robotic or manual handlingmethods can probe chemical interactions and determine levels ofexpression of many genes in a short period of time. Techniques have beendeveloped that utilize molecular signals (e.g., fluorophores) andautomated analyses that process information at a very rapid rate (see,e.g., Pinhasov et al., Comb. Chem. High Throughput Screen. 7:133(2004)). For example, microarray technology has been extensivelyutilized to probe the interactions of thousands of genes at once, whileproviding information for specific genes (see, e.g., Mocellin and Rossi,Adv. Exp. Med. Biol. 593:19 (2007)).

Such screening methods (e.g., high-throughput) can identify activeagents and compounds rapidly and efficiently. For example, cells can bepositioned or placed (pre-seeded) on a culture dish, tube, flask, rollerbottle or plate (e.g., a single multi-well plate or dish such as an 8,16, 32, 64, 96, 384 and 1536 multi-well plate or dish), optionally atdefined locations, for identification of potentially therapeuticmolecules. Libraries that can be screened include, for example, smallmolecule libraries, phage display libraries, fully human antibody yeastdisplay libraries (Adimab, LLC), siRNA libraries, and adenoviraltransfection vectors.

XIII. Pharmaceutical Preparations and Therapeutic Uses

a. Formulations and Routes of Administration

Depending on the form of the modulator along with any optionalconjugate, the mode of intended delivery, the disease being treated ormonitored and numerous other variables, compositions of the instantinvention may be formulated as desired using art recognized techniques.That is, in various embodiments of the instant invention compositionscomprising EFNA modulators are formulated with a wide variety ofpharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: TheScience and Practice of Pharmacy with Facts and Comparisons: DrugfactsPlus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms andDrug Delivery Systems, 7^(th) ed., Lippencott Williams and Wilkins(2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3^(rd) ed.,Pharmaceutical Press (2000)). Various pharmaceutically acceptablecarriers, which include vehicles, adjuvants, and diluents, are readilyavailable from numerous commercial sources. Moreover, an assortment ofpharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are also available. Certain non-limiting exemplarycarriers include saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof.

More particularly it will be appreciated that, in some embodiments, thetherapeutic compositions of the invention may be administered neat orwith a minimum of additional components. Conversely the EFNA modulatorsof the present invention may optionally be formulated to containsuitable pharmaceutically acceptable carriers comprising excipients andauxiliaries that are well known in the art and are relatively inertsubstances that facilitate administration of the modulator or which aidprocessing of the active compounds into preparations that arepharmaceutically optimized for delivery to the site of action. Forexample, an excipient can give form or consistency or act as a diluentto improve the pharmacokinetics of the modulator. Suitable excipientsinclude but are not limited to stabilizing agents, wetting andemulsifying agents, salts for varying osmolarity, encapsulating agents,buffers, and skin penetration enhancers.

Disclosed modulators for systemic administration may be formulated forenteral, parenteral or topical administration. Indeed, all three typesof formulation may be used simultaneously to achieve systemicadministration of the active ingredient. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing (2000). Suitable formulations for parenteral administrationinclude aqueous solutions of the active compounds in water-soluble form,for example, water-soluble salts. In addition, suspensions of the activecompounds as appropriate for oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, ethyl oleate or triglycerides. Aqueous injection suspensionsmay contain substances that increase the viscosity of the suspension andinclude, for example, sodium carboxymethyl cellulose, sorbitol, and/ordextran. Optionally, the suspension may also contain stabilizers.Liposomes can also be used to encapsulate the agent for delivery intothe cell.

Suitable formulations for enteral administration include hard or softgelatin capsules, pills, tablets, including coated tablets, elixirs,suspensions, syrups or inhalations and controlled release forms thereof.

In general the compounds and compositions of the invention, comprisingEFNA modulators may be administered in vivo, to a subject in needthereof, by various routes, including, but not limited to, oral,intravenous, intra-arterial, subcutaneous, parenteral, intranasal,intramuscular, intracardiac, intraventricular, intratracheal, buccal,rectal, intraperitoneal, intradermal, topical, transdermal, andintrathecal, or otherwise by implantation or inhalation. The subjectcompositions may be formulated into preparations in solid, semi-solid,liquid, or gaseous forms; including, but not limited to, tablets,capsules, powders, granules, ointments, solutions, suppositories,enemas, injections, inhalants, and aerosols. The appropriate formulationand route of administration may be selected according to the intendedapplication and therapeutic regimen.

b. Dosages

Similarly, the particular dosage regimen, i.e., dose, timing andrepetition, will depend on the particular individual and thatindividual's medical history. Empirical considerations such aspharmacokinetics (e.g., half-life, clearance rate, etc.) will contributeto the determination of the dosage. Frequency of administration may bedetermined and adjusted over the course of therapy, and is based onreducing the number of hyperproliferative or neoplastic cells, includingtumor initiating cells, maintaining the reduction of such neoplasticcells, reducing the proliferation of neoplastic cells, or delaying thedevelopment of metastasis. Alternatively, sustained continuous releaseformulations of a subject therapeutic composition may be appropriate. Asalluded to above various formulations and devices for achievingsustained release are known in the art.

From a therapeutic standpoint the pharmaceutical compositions areadministered in an amount effective for treatment or prophylaxis of thespecific indication. The therapeutically effective amount is typicallydependent on the weight of the subject being treated, his or herphysical or health condition, the extensiveness of the condition to betreated, or the age of the subject being treated. In general, the EFNAmodulators of the invention may be administered in an amount in therange of about 10 μg/kg body weight to about 100 mg/kg body weight perdose. In certain embodiments, the EFNA modulators of the invention maybe administered in an amount in the range of about 50 μg/kg body weightto about 5 mg/kg body weight per dose. In certain other embodiments, theEFNA modulators of the invention may be administered in an amount in therange of about 100 μg/kg body weight to about 10 mg/kg body weight perdose. Optionally, the EFNA modulators of the invention may beadministered in an amount in the range of about 100 μg/kg body weight toabout 20 mg/kg body weight per dose. Further optionally, the EFNAmodulators of the invention may be administered in an amount in therange of about 0.5 mg/kg body weight to about 20 mg/kg body weight perdose. In certain embodiments the compounds of present invention areprovided a dose of at least about 100 pg/kg body weight, at least about250 μg/kg body weight, at least about 750 μg/kg body weight, at leastabout 3 mg/kg body weight, at least about 5 mg/kg body weight, at leastabout 10 mg/kg body weight is administered.

Other dosing regimens may be predicated on Body Surface Area (BSA)calculations as disclosed in U.S. Pat. No. 7,744,877 which isincorporated herein by reference in its entirety. As is well known inthe art the BSA is calculated using the patient's height and weight andprovides a measure of a subject's size as represented by the surfacearea of his or her body. In selected embodiments of the invention usingthe BSA the modulators may be administered in dosages from 10 mg/m² to800 mg/m². In other preferred embodiments the modulators will beadministered in dosages from 50 mg/m² to 500 mg/m² and even mg/m², 350mg/m², 400 mg/m² or 450 mg/m². Of course it will be appreciated that,regardless of how the dosages are calculated, multiple dosages may beadministered over a selected time period to provide an absolute dosagethat is substantially higher than the individual administrations.

In any event, the EFNA modulators are preferably administered as neededto subjects in need thereof. Determination of the frequency ofadministration may be made by persons skilled in the art, such as anattending physician based on considerations of the condition beingtreated, age of the subject being treated, severity of the conditionbeing treated, general state of health of the subject being treated andthe like. Generally, an effective dose of the EFNA modulator isadministered to a subject one or more times. More particularly, aneffective dose of the modulator is administered to the subject once amonth, more than once a month, or less than once a month. In certainembodiments, the effective dose of the EFNA modulator may beadministered multiple times, including for periods of at least a month,at least six months, or at least a year.

Dosages and regimens may also be determined empirically for thedisclosed therapeutic compositions in individuals who have been givenone or more administration(s). For example, individuals may be givenincremental dosages of a therapeutic composition produced as describedherein. To assess efficacy of the selected composition, a marker of thespecific disease, disorder or condition can be followed as describedpreviously. In embodiments where the individual has cancer, theseinclude direct measurements of tumor size via palpation or visualobservation, indirect measurement of tumor size by x-ray or otherimaging techniques; an improvement as assessed by direct tumor biopsyand microscopic examination of the tumor sample; the measurement of anindirect tumor marker (e.g., PSA for prostate cancer) or an antigenidentified according to the methods described herein, a decrease in painor paralysis; improved speech, vision, breathing or other disabilityassociated with the tumor; increased appetite; or an increase in qualityof life as measured by accepted tests or prolongation of survival. Itwill be apparent to one of skill in the art that the dosage will varydepending on the individual, the type of neoplastic condition, the stageof neoplastic condition, whether the neoplastic condition has begun tometastasize to other location in the individual, and the past andconcurrent treatments being used.

c. Combination Therapies

Combination therapies contemplated by the invention may be particularlyuseful in decreasing or inhibiting unwanted neoplastic cellproliferation (e.g. endothelial cells), decreasing the occurrence ofcancer, decreasing or preventing the recurrence of cancer, or decreasingor preventing the spread or metastasis of cancer. In such cases thecompounds of the instant invention may function as sensitizing orchemosensitizing agent by removing the TPC propping up and perpetuatingthe tumor mass (e.g. NTG cells) and allow for more effective use ofcurrent standard of care debulking or anti-cancer agents. That is, acombination therapy comprising an EFNA modulator and one or moreanti-cancer agents may be used to diminish established cancer e.g.,decrease the number of cancer cells present and/or decrease tumorburden, or ameliorate at least one manifestation or side effect ofcancer. As such, combination therapy refers to the administration of aEFNA modulator and one or more anti-cancer agent that include, but arenot limited to, cytotoxic agents, cytostatic agents, chemotherapeuticagents, targeted anti-cancer agents, biological response modifiers,immunotherapeutic agents, cancer vaccines, anti-angiogenic agents,cytokines, hormone therapies, radiation therapy and anti-metastaticagents.

According to the methods of the present invention, there is norequirement for the combined results to be additive of the effectsobserved when each treatment (e.g., anti-EFNA antibody and anti-canceragent) is conducted separately. Although at least additive effects aregenerally desirable, any increased anti-tumor effect above one of thesingle therapies is beneficial. Furthermore, the invention does notrequire the combined treatment to exhibit synergistic effects. However,those skilled in the art will appreciate that with certain selectedcombinations that comprise preferred embodiments, synergism may beobserved.

To practice combination therapy according to the invention, an EFNAmodulator (e.g., anti-EFNA antibody) in combination with one or moreanti-cancer agent may be administered to a subject in need thereof in amanner effective to result in anti-cancer activity within the subject.The EFNA modulator and anti-cancer agent are provided in amountseffective and for periods of time effective to result in their combinedpresence and their combined actions in the tumor environment as desired.To achieve this goal, the EFNA modulator and anti-cancer agent may beadministered to the subject simultaneously, either in a singlecomposition, or as two or more distinct compositions using the same ordifferent administration routes.

Alternatively, the modulator may precede, or follow, the anti-canceragent treatment by, e.g., intervals ranging from minutes to weeks. Incertain embodiments wherein the anti-cancer agent and the antibody areapplied separately to the subject, the time period between the time ofeach delivery is such that the anti-cancer agent and modulator are ableto exert a combined effect on the tumor. In a particular embodiment, itis contemplated that both the anti-cancer agent and the EFNA modulatorare administered within about 5 minutes to about two weeks of eachother.

In yet other embodiments, several days (2, 3, 4, 5, 6 or 7), severalweeks (1, 2, 3, 4, 5, 6, 7 or 8) or several months (1, 2, 3, 4, 5, 6, 7or 8) may lapse between administration of the modulator and theanti-cancer agent. The EFNA modulator and one or more anti-cancer agent(combination therapy) may be administered once, twice or at least theperiod of time until the condition is treated, palliated or cured.Preferably, the combination therapy is administered multiple times. Thecombination therapy may be administered from three times daily to onceevery six months. The administering may be on a schedule such as threetimes daily, twice daily, once daily, once every two days, once everythree days, once weekly, once every two weeks, once every month, onceevery two months, once every three months, once every six months or maybe administered continuously via a minipump. As previously indicated thecombination therapy may be administered via an oral, mucosal, buccal,intranasal, inhalable, intravenous, subcutaneous, intramuscular,parenteral, intratumor or topical route. The combination therapy may beadministered at a site distant from the site of the tumor. Thecombination therapy generally will be administered for as long as thetumor is present provided that the combination therapy causes the tumoror cancer to stop growing or to decrease in weight or volume.

In one embodiment an EFNA modulator is administered in combination withone or more anti-cancer agents for a short treatment cycle to a subjectin need thereof. The duration of treatment with the antibody may varyaccording to the particular anti-cancer agent used. The invention alsocontemplates discontinuous administration or daily doses divided intoseveral partial administrations. An appropriate treatment time for aparticular anti-cancer agent will be appreciated by the skilled artisan,and the invention contemplates the continued assessment of optimaltreatment schedules for each anti-cancer agent.

The present invention contemplates at least one cycle, preferably morethan one cycle during which the combination therapy is administered. Anappropriate period of time for one cycle will be appreciated by theskilled artisan, as will the total number of cycles, and the intervalbetween cycles. The invention contemplates the continued assessment ofoptimal treatment schedules for each modulator and anti-cancer agent.Moreover, the invention also provides for more than one administrationof either the anti-EFNA antibody or the anti-cancer agent. The modulatorand anti-cancer agent may be administered interchangeably, on alternatedays or weeks; or a sequence of antibody treatment may be given,followed by one or more treatments of anti-cancer agent therapy. In anyevent, as will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will be generally aroundthose already employed in clinical therapies wherein thechemotherapeutics are administered alone or in combination with otherchemotherapeutics.

In another preferred embodiment the EFNA modulators of the instantinvention may be used in maintenance therapy to reduce or eliminate thechance of tumor recurrence following the initial presentation of thedisease. Preferably the disorder will have been treated and the initialtumor mass eliminated, reduced or otherwise ameliorated so the patientis asymptomatic or in remission. As such time the subject may beadministered pharmaceutically effective amounts of the disclosedmodulators one or more times even though there is little or noindication of disease using standard diagnostic procedures. In someembodiments the effectors will be administered on a regular scheduleover a period of time. For example the EFNA modulators could beadministered weekly, every two weeks, monthly, every six weeks, everytwo months, every three months every six months or annually. Given theteachings herein, one skilled in the art could readily determinefavorable dosages and dosing regimens to reduce the potential of diseaserecurrence. Moreover such treatments could be continued for a period ofweeks, months, years or even indefinitely depending on the patientresponse and clinical and diagnostic parameters.

In yet another preferred embodiment the effectors of the presentinvention may be used to prophylactically to prevent or reduce thepossibility of tumor metastasis following a debulking procedure. As usedin the instant disclosure a debulking procedure is defined broadly andshall mean any procedure, technique or method that eliminates, reduces,treats or ameliorates a tumor or tumor proliferation. Exemplarydebulking procedures include, but are not limited to, surgery, radiationtreatments (i.e., beam radiation), chemotherapy or ablation. Atappropriate times readily determined by one skilled in the art in viewof the instant disclosure the EFNA modulators may be administered assuggested by clinical and diagnostic or theragnostic procedures toreduce tumor metastasis. The modulators may be administered one or moretimes at pharmaceutically effective dosages as determined using standardtechniques. Preferably the dosing regimen will be accompanied byappropriate diagnostic or monitoring techniques that allow it to bemodified as necessary.

d. Anti-Cancer Agents

As used herein the term anti-cancer agent means any agent that can beused to treat a cell proliferative disorder such as cancer, includingcytotoxic agents, cytostatic agents, anti-angiogenic agents, debulkingagents, chemotherapeutic agents, radiotherapy and radiotherapeuticagents, targeted anti-cancer agents, biological response modifiers,antibodies, and immunotherapeutic agents. It will be appreciated that,in selected embodiments as discussed above, anti-cancer agents maycomprise conjugates and may be associated with modulators prior toadministration.

The term cytotoxic agent means a substance that decreases or inhibitsthe function of cells and/or causes destruction of cells, i.e., thesubstance is toxic to the cells. Typically, the substance is a naturallyoccurring molecule derived from a living organism. Examples of cytotoxicagents include, but are not limited to, small molecule toxins orenzymatically active toxins of bacteria (e.g., Diptheria toxin,Pseudomonas endotoxin and exotoxin, Staphylococcal enterotoxin A),fungal (e.g., α-sarcin, restrictocin), plants (e.g., abrin, ricin,modeccin, viscumin, pokeweed anti-viral protein, saporin, gelonin,momoridin, trichosanthin, barley toxin, Aleurites fordii proteins,dianthin proteins, Phytolacca mericana proteins (PAPI, PAPII, andPAP-S), Momordica charantia inhibitor, curcin, crotin, saponariaofficinalis inhibitor, gelonin, mitegellin, restrictocin, phenomycin,neomycin, and the tricothecenes) or animals, e.g., cytotoxic RNases,such as extracellular pancreatic RNases; DNase I, including fragmentsand/or variants thereof.

A chemotherapeutic agent means a chemical compound that non-specificallydecreases or inhibits the growth, proliferation, and/or survival ofcancer cells (e.g., cytotoxic or cytostatic agents). Such chemicalagents are often directed to intracellular processes necessary for cellgrowth or division, and are thus particularly effective againstcancerous cells, which generally grow and divide rapidly. For example,vincristine depolymerizes microtubules, and thus inhibits cells fromentering mitosis. In general, chemotherapeutic agents can include anychemical agent that inhibits, or is designed to inhibit, a cancerouscell or a cell likely to become cancerous or generate tumorigenicprogeny (e.g., TIC). Such agents are often administered, and are oftenmost effective, in combination, e.g., in the formulation CHOP.

Examples of anti-cancer agents that may be used in combination with (orconjugated to) the modulators of the present invention include, but arenot limited to, alkylating agents, alkyl sulfonates, aziridines,ethylenimines and methylamelamines, acetogenins, a camptothecin,bryostatin, callystatin, CC-1065, cryptophycins, dolastatin,duocarmycin, eleutherobin, pancratistatin, a sarcodictyin, spongistatin,nitrogen mustards, antibiotics, enediyne antibiotics, dynemicin,bisphosphonates, an esperamicin, chromoprotein enediyne antiobioticchromophores, aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin,chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin, epirubicin,esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid,nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites, folic acid analogues,purine analogs, androgens, anti-adrenals, folic acid replenisher such asfrolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinicacid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate,defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate,an epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan,lonidainine, maytansinoids, mitoguazone, mitoxantrone, mopidanmol,nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone,podophyllinic acid, 2-ethylhydrazide, procarbazine, PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.), razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids,chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine;methotrexate; platinum analogs, vinblastine; platinum; etoposide(VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine;novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda;ibandronate; irinotecan (Camptosar, CPT-11), topoisomerase inhibitor RFS2000; difluorometlhylornithine (DMFO); retinoids; capecitabine;combretastatin; leucovorin (LV); oxaliplatin; inhibitors of PKC-alpha,Raf, H-Ras, EGFR and VEGF-A that reduce cell proliferation andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Also included in this definition are anti-hormonal agents thatact to regulate or inhibit hormone action on tumors such asanti-estrogens and selective estrogen receptor modulators (SERMs),aromatase inhibitors that inhibit the enzyme aromatase, which regulatesestrogen production in the adrenal glands, and anti-androgens; as wellas troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides; ribozymes such as a VEGF expression inhibitor and aHER2 expression inhibitor; vaccines, PROLEUKIN® rIL-2; LURTOTECAN®topoisomerase 1 inhibitor; ABARELIX® rmRH; Vinorelbine and Esperamicinsand pharmaceutically acceptable salts, acids or derivatives of any ofthe above. Other embodiments comprise the use of antibodies approved forcancer therapy including, but not limited to, rituximab, trastuzumab,gemtuzumab ozogamcin, alemtuzumab, ibritumomab tiuxetan, tositumomab,bevacizumab, cetuximab, patitumumab, ofatumumab, ipilimumab andbrentuximab vedotin. Those skilled in the art will be able to readilyidentify additional anti-cancer agents that are compatible with theteachings herein.

e. Radiotherapy

The present invention also provides for the combination of EFNAmodulators with radiotherapy (i.e., any mechanism for inducing DNAdamage locally within tumor cells such as gamma.-irradiation, X-rays,UV-irradiation, microwaves, electronic emissions and the like).Combination therapy using the directed delivery of radioisotopes totumor cells is also contemplated, and may be used in connection with atargeted anti-cancer agent or other targeting means. Typically,radiation therapy is administered in pulses over a period of time fromabout 1 to about 2 weeks. The radiation therapy may be administered tosubjects having head and neck cancer for about 6 to 7 weeks. Optionally,the radiation therapy may be administered as a single dose or asmultiple, sequential doses.

f. Neoplastic Conditions

Whether administered alone or in combination with an anti-cancer agentor radiotherapy, the EFNA modulators of the instant invention areparticularly useful for generally treating neoplastic conditions inpatients or subjects which may include benign or malignant tumors (e.g.,renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal,prostate, pancreatic, lung, thyroid, hepatic carcinomas; sarcomas;glioblastomas; and various head and neck tumors); leukemias and lymphoidmalignancies; other disorders such as neuronal, glial, astrocytal,hypothalamic and other glandular, macrophagal, epithelial, stromal andblastocoelic disorders; and inflammatory, angiogenic, immunologicdisorders and disorders caused by pathogens. Particularly preferredtargets for treatment with therapeutic compositions and methods of thepresent invention are neoplastic conditions comprising solid tumors. Inother preferred embodiments the modulators of the present invention maybe used for the diagnosis, prevention or treatment of hematologicmalignancies. Preferably the subject or patient to be treated will behuman although, as used herein, the terms are expressly held to compriseany mammalian species.

More specifically, neoplastic conditions subject to treatment inaccordance with the instant invention may be selected from the groupincluding, but not limited to, adrenal gland tumors, AIDS-associatedcancers, alveolar soft part sarcoma, astrocytic tumors, bladder cancer(squamous cell carcinoma and transitional cell carcinoma), bone cancer(adamantinoma, aneurism al bone cysts, osteochondroma, osteosarcoma),brain and spinal cord cancers, metastatic brain tumors, breast cancer,carotid body tumors, cervical cancer, chondrosarcoma, chordoma,chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer,colorectal cancer, cutaneous benign fibrous histiocytomas, desmoplasticsmall round cell tumors, ependymomas, Ewing's tumors, extraskeletalmyxoid chondrosarcoma, fibrogenesis imperfecta ossium, fibrous dysplasiaof the bone, gallbladder and bile duct cancers, gestationaltrophoblastic disease, germ cell tumors, head and neck cancers, isletcell tumors, Kaposi's Sarcoma, kidney cancer (nephroblastoma, papillaryrenal cell carcinoma), leukemias, lipoma/benign lipomatous tumors,liposarcoma/malignant lipomatous tumors, liver cancer (hepatoblastoma,hepatocellular carcinoma), lymphomas, lung cancers (small cellcarcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinomaetc.), medulloblastoma, melanoma, meningiomas, multiple endocrineneoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma,neuroendocrine tumors, ovarian cancer, pancreatic cancers, papillarythyroid carcinomas, parathyroid tumors, pediatric cancers, peripheralnerve sheath tumors, phaeochromocytoma, pituitary tumors, prostatecancer, posterious unveal melanoma, rare hematologic disorders, renalmetastatic cancer, rhabdoid tumor, rhabdomysarcoma, sarcomas, skincancer, soft-tissue sarcomas, squamous cell cancer, stomach cancer,synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroidmetastatic cancer, and uterine cancers (carcinoma of the cervix,endometrial carcinoma, and Ieiomyoma). In certain preferred embodiments,the cancerous cells are selected from the group of solid tumorsincluding but not limited to breast cancer, non-small cell lung cancer(NSCLC), small cell lung cancer, pancreatic cancer, colon cancer,prostate cancer, sarcomas, renal metastatic cancer, thyroid metastaticcancer, and clear cell carcinoma.

With regard to hematologic malignancies it will be further beappreciated that the compounds and methods of the present invention maybe particularly effective in treating a variety of B-cell lymphomas,including low grade/NHL follicular cell lymphoma (FCC), mantle celllymphoma (MCL), diffuse large cell lymphoma (DLCL), small lymphocytic(SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuseNHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, highgrade small non-cleaved cell NHL, bulky disease NHL, Waldenstrom'sMacroglobulinemia, lymphoplasmacytoid lymphoma (LPL), mantle celllymphoma (MCL), follicular lymphoma (FL), diffuse large cell lymphoma(DLCL), Burkitt's lymphoma (BL), AIDS-related lymphomas, monocytic Bcell lymphoma, angioimmunoblastic lymphoadenopathy, small lymphocytic,follicular, diffuse large cell, diffuse small cleaved cell, large cellimmunoblastic lymphoblastoma, small, non-cleaved, Burkitt's andnon-Burkitt's, follicular, predominantly large cell; follicular,predominantly small cleaved cell; and follicular, mixed small cleavedand large cell lymphomas. See, Gaidono et al., “Lymphomas”, IN CANCER:PRINCIPLES & PRACTICE OF ONCOLOGY, Vol. 2: 2131-2145 (DeVita et al.,eds., 5.sup.th ed. 1997). It should be clear to those of skill in theart that these lymphomas will often have different names due to changingsystems of classification, and that patients having lymphomas classifiedunder different names may also benefit from the combined therapeuticregimens of the present invention.

In yet other preferred embodiments the EFNA modulators may be used toeffectively treat certain myeloid and hematologic malignancies includingleukemias such as chronic lymphocytic leukemia (CLL or B-CLL). CLL ispredominantly a disease of the elderly that starts to increase inincidence after fifty years of age and reaches a peak by late sixties.It generally involves the proliferation of neoplastic peripheral bloodlymphocytes. Clinical finding of CLL involves lymphocytosis,lymphadenopatliy, splenomegaly, anemia and thrombocytopenia. Acharacteristic feature of CLL is monoclonal B cell proliferation andaccumulation of B-lymphocytes arrested at an intermediate state ofdifferentiation where such B cells express surface IgM (sIgM) or bothsIgM and sIgD, and a single light chain at densities lower than that onthe normal B cells. However, as discussed above and shown in theExamples appended hereto, selected EFNA expression (e.g., EFNA) isupregulated on B-CLL cells thereby providing an attractive target forthe disclosed modulators.

The present invention also provides for a preventative or prophylactictreatment of subjects who present with benign or precancerous tumors. Itis not believed that any particular type of tumor or neoplastic disordershould be excluded from treatment using the present invention. However,the type of tumor cells may be relevant to the use of the invention incombination with secondary therapeutic agents, particularlychemotherapeutic agents and targeted anti-cancer agents.

Still other preferred embodiments of the instant invention comprise theuse of EFNA modulators to treat subjects suffering from solid tumors. Insuch subjects many of these solid tumors comprise tissue exhibitingvarious genetic mutations that may render them particularly susceptibleto treatment with the disclosed effectors. For example, KRAS, APC andCTNNB1 and CDH1 mutations are relatively common in patients withcolorectal cancer. Moreover, patients suffering from tumors with thesemutations are usually the most refractory to current therapies;especially those patients with KRAS mutations. KRAS activatingmutations, which typically result in single amino acid substitutions,are also implicated in other difficult to treat malignancies, includinglung adenocarcinoma, mucinous adenoma, and ductal carcinoma of thepancreas.

Currently, the most reliable prediction of whether colorectal cancerpatients will respond to EGFR- or VEGF-inhibiting drugs, for example, isto test for certain KRAS “activating” mutations. KRAS is mutated in35-45% of colorectal cancers, and patients whose tumors express mutatedKRAS do not respond well to these drugs. For example, KRAS mutations arepredictive of a lack of response to panitumumab and cetuximab therapy incolorectal cancer (Lievre et al. Cancer Res 66:3992-5; Karapetis et al.NEJM 359:1757-1765). Approximately 85% of patients with colorectalcancer have mutations in the APC gene (Markowitz & Bertagnolli. NEJM361:2449-60), and more than 800 APC mutations have been characterized inpatients with familial adenomatous polyposis and colorectal cancer. Amajority of these mutations result in a truncated APC protein withreduced functional ability to mediate the destruction of beta-catenin.Mutations in the beta-catenin gene, CTNNB1, can also result in increasedstabilization of the protein, resulting in nuclear import and subsequentactivation of several oncogenic transcriptional programs, which is alsothe mechanism of oncogenesis resulting from failure of mutated APC toappropriately mediate beta-catenin destruction, which is required tokeep normal cell proliferation and differentiation programs in check.

Loss of CDH1 (E-cadherin) expression is yet another common occurrence incolorectal cancer, often observed in more advanced stages of thedisease. E-cadherin is the central member of adherin junctions thatconnect and organize cells in epithelial layers. Normally E-cadherinphysically sequesters beta-catenin (CTNNB1) at the plasma membrane; lossof E-cadherin expression in colorectal cancer results in localization ofbeta-catenin to the nucleus and transcriptional activation of thebeta-catenin/WNT pathway. Aberrant beta-catenin/WNT signaling is centralto oncogenesis and nuclear beta-catenin has been implicated in cancerstemness (Schmalhofer et al., 2009 PMID 19153669). E-cadherin isrequired for the expression and function of EphA2 a known bindingpartner for EFNA ligands in epithelia cells (Dodge Zantek et al., 1999PMID 10511313; Orsulic S and Kemler R, 2000 PMID 10769210). Usingmodulators that bind to EFNA ligands and agonize with or antagonize Ephreceptor binding may modify, interrupt or revert pro-oncogenicprocesses. Alternatively, EFNA modulators may preferentially bind totumor cells with aberrant Eph/ephrin interactions based on the bindingpreferences of the EFNA modulators. Hence patients with cancers carryingthe above mentioned genetic traits may benefits from treatment withaforementioned EFNA modulators.

XIV. Articles of Manufacture

Pharmaceutical packs and kits comprising one or more containers,comprising one or more doses of an EFNA modulator are also provided. Incertain embodiments, a unit dosage is provided wherein the unit dosagecontains a predetermined amount of a composition comprising, forexample, an anti-EFNA antibody, with or without one or more additionalagents. For other embodiments, such a unit dosage is supplied insingle-use prefilled syringe for injection. In still other embodiments,the composition contained in the unit dosage may comprise saline,sucrose, or the like; a buffer, such as phosphate, or the like; and/orbe formulated within a stable and effective pH range. Alternatively, incertain embodiments, the composition may be provided as a lyophilizedpowder that may be reconstituted upon addition of an appropriate liquid,for example, sterile water. In certain preferred embodiments, thecomposition comprises one or more substances that inhibit proteinaggregation, including, but not limited to, sucrose and arginine. Anylabel on, or associated with, the container(s) indicates that theenclosed composition is used for diagnosing or treating the diseasecondition of choice.

The present invention also provides kits for producing single-dose ormulti-dose administration units of an EFNA modulator and, optionally,one or more anti-cancer agents. The kit comprises a container and alabel or package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, etc. Thecontainers may be formed from a variety of materials such as glass orplastic. The container holds a composition that is effective fortreating the condition and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). Such kits willgenerally contain in a suitable container a pharmaceutically acceptableformulation of the EFNA modulator and, optionally, one or moreanti-cancer agents in the same or different containers. The kits mayalso contain other pharmaceutically acceptable formulations, either fordiagnosis or combined therapy. For example, in addition to the EFNAmodulator of the invention such kits may contain any one or more of arange of anti-cancer agents such as chemotherapeutic or radiotherapeuticdrugs; anti-angiogenic agents; anti-metastatic agents; targetedanti-cancer agents; cytotoxic agents; and/or other anti-cancer agents.Such kits may also provide appropriate reagents to conjugate the EFNAmodulator with an anti-cancer agent or diagnostic agent (e.g., see U.S.Pat. No. 7,422,739 which is incorporated herein by reference in itsentirety).

More specifically the kits may have a single container that contains theEFNA modulator, with or without additional components, or they may havedistinct containers for each desired agent. Where combined therapeuticsare provided for conjugation, a single solution may be pre-mixed, eitherin a molar equivalent combination, or with one component in excess ofthe other. Alternatively, the EFNA modulator and any optionalanti-cancer agent of the kit may be maintained separately withindistinct containers prior to administration to a patient. The kits mayalso comprise a second/third container means for containing a sterile,pharmaceutically acceptable buffer or other diluent such asbacteriostatic water for injection (BWFI), phosphate-buffered saline(PBS), Ringer's solution and dextrose solution.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution is preferably an aqueous solution, with asterile aqueous solution being particularly preferred. However, thecomponents of the kit may be provided as dried powder(s). When reagentsor components are provided as a dry powder, the powder can bereconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container.

As indicated briefly above the kits may also contain a means by which toadminister the antibody and any optional components to an animal orpatient, e.g., one or more needles or syringes, or even an eye dropper,pipette, or other such like apparatus, from which the formulation may beinjected or introduced into the animal or applied to a diseased area ofthe body. The kits of the present invention will also typically includea means for containing the vials, or such like, and other component inclose confinement for commercial sale, such as, e.g., injection orblow-molded plastic containers into which the desired vials and otherapparatus are placed and retained. Any label or package insert indicatesthat the EFNA modulator composition is used for treating cancer, forexample colorectal cancer.

XV. Research Reagents

Other preferred embodiments of the invention also exploit the propertiesof the disclosed modulators as an instrument useful for identifying,isolating, sectioning or enriching populations or subpopulations oftumor initiating cells through methods such as fluorescent activatedcell sorting (FACS), magnetic activated cell sorting (MACS) or lasermediated sectioning. Those skilled in the art will appreciate that themodulators may be used in several compatible techniques for thecharacterization and manipulation of TIC including cancer stem cells(e.g., see U.S. Ser. Nos. 12/686,359, 12/669,136 and 12/757,649 each ofwhich is incorporated herein by reference in its entirety).

XVI. Miscellaneous

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Morespecifically, as used in this specification and the appended claims, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aprotein” includes a plurality of proteins; reference to “a cell”includes mixtures of cells, and the like. In addition, ranges providedin the specification and appended claims include both end points and allpoints between the end points. Therefore, a range of 2.0 to 3.0 includes2.0, 3.0, and all points between 2.0 and 3.0.

Generally, nomenclature used in connection with, and techniques of, celland tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook J. & Russell D. Molecular Cloning: ALaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2000); Ausubel et al., Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, Wiley, John & Sons, Inc. (2002); Harlow and Lane UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols inProtein Science, Wiley, John & Sons, Inc. (2003). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications, as commonly accomplished in the art or as describedherein. The nomenclature used in connection with, and the laboratoryprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art.

All references or documents disclosed or cited within this specificationare, without limitation, incorporated herein by reference in theirentirety. Moreover, any section headings used herein are fororganizational purposes only and are not to be construed as limiting thesubject matter described.

EXAMPLES

The present invention, thus generally described above, will beunderstood more readily by reference to the following examples, whichare provided by way of illustration and are not intended to be limitingof the instant invention. The examples are not intended to representthat the experiments below are all or the only experiments performed.Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Example 1 Enrichment of Tumor Initiating Cell Populations

To characterize the cellular heterogeneity of solid tumors as they existin cancer patients, elucidate the identity of tumor perpetuating cells(TPC; i.e. cancer stem cells: CSC) using particular phenotypic markersand identify clinically relevant therapeutic targets, a largenon-traditional xenograft (NTX) tumor bank was developed and maintainedusing art recognized techniques. The NTX tumor bank, comprising a largenumber of discrete tumor cell lines, was propagated in immunocompromisedmice through multiple passages of heterogeneous tumor cells originallyobtained from numerous cancer patients afflicted by a variety of solidtumor malignancies. The continued availability of a large number ofdiscrete early passage NTX tumor cell lines having well defined lineagesgreatly facilitate the identification and isolation of TPC as they allowfor the reproducible and repeated characterization of cells purifiedfrom the cell lines. More particularly, isolated or purified TPC aremost accurately defined retrospectively according to their ability togenerate phenotypically and morphologically heterogeneous tumors in micethat recapitulate the patient tumor sample from which the cellsoriginated. Thus, the ability to use small populations of isolated cellsto generate fully heterogeneous tumors in mice is strongly indicative ofthe fact that the isolated cells comprise TPC. In such work the use ofminimally passaged NTX cell lines greatly simplifies in vivoexperimentation and provides readily verifiable results. Moreover, earlypassage NTX tumors also respond to therapeutic agents such as irinotecan(i.e. Camptosar®), which provides clinically relevant insights intounderlying mechanisms driving tumor growth, resistance to currenttherapies and tumor recurrence.

As the NTX tumor cell lines were established the constituent tumor cellphenotypes were analyzed using flow cytometry to identify discretemarkers that might be used to characterize, isolate, purify or enrichtumor initiating cells (TIC) and separate or analyze TPC and TProg cellswithin such populations. In this regard the inventors employed aproprietary proteomic based platform (i.e. PhenoPrint™ Array) thatprovided for the rapid characterization of cells based on proteinexpression and the concomitant identification of potentially usefulmarkers. The PhenoPrint Array is a proprietary proteomic platformcomprising hundreds of discrete binding molecules, many obtained fromcommercial sources, arrayed in 96 well plates wherein each well containsa distinct antibody in the phycoerythrin fluorescent channel andmultiple additional antibodies in different fluorochromes arrayed inevery well across the plate. This allows for the determination ofexpression levels of the antigen of interest in a subpopulation ofselected tumor cells through rapid inclusion of relevant cells orelimination of non-relevant cells via non-phycoerythrin channels. Whenthe PhenoPrint Array was used in combination with tissue dissociation,transplantation and stem cell techniques well known in the art (Al-Hajjet al., 2004, Dalerba et al., 2007 and Dylla et al., 2008, all supra,each of which is incorporated herein by reference in its entirety), itwas possible to effectively identify relevant markers and subsequentlyisolate and transplant specific human tumor cell subpopulations withgreat efficiency.

Accordingly, upon establishing various NTX tumor cell lines as iscommonly done for human tumors in severely immune compromised mice, thetumors were resected from mice upon reaching 800-2,000 mm³ and the cellswere dissociated into single cell suspensions using art-recognizedenzymatic digestion techniques (See for example U.S.P.N. 2007/0292414which is incorporated herein). Data obtained from these suspensionsusing the PhenoPrint Array provided both absolute (per cell) andrelative (vs. other cells in the population) surface protein expressionon a cell-by-cell basis, leading to more complex characterization andstratification of cell populations. More specifically, use of thePhenoPrint Array allowed for the rapid identification of proteins ormarkers that prospectively distinguished TIC or TPC from NTG bulk tumorcells and tumor stroma and, when isolated from NTX tumor models,provided for the relatively rapid characterization of tumor cellsubpopulations expressing differing levels of specific cell surfaceproteins. In particular, proteins with heterogeneous expression acrossthe tumor cell population allow for the isolation and transplantation ofdistinct, and highly purified, tumor cell subpopulations expressingeither high and low levels of a particular protein or marker intoimmune-compromised mice, thereby facilitating the assessment of whetherTPC were enriched in one subpopulation or another.

The term enriching is used synonymously with isolating cells and meansthat the yield (fraction) of cells of one type is increased over thefraction of other types of cells as compared to the starting or initialcell population. Preferably, enriching refers to increasing thepercentage by about 10%, by about 20%, by about 30%, by about 40%, byabout 50% or greater than 50% of one type of cell in a population ofcells as compared to the starting population of cells.

As used herein a marker, in the context of a cell or tissue, means anycharacteristic in the form of a chemical or biological entity that isidentifiably associated with, or specifically found in or on aparticular cell, cell population or tissue including those identified inor on a tissue or cell population affected by a disease or disorder. Asmanifested, markers may be morphological, functional or biochemical innature. In preferred embodiments the marker is a cell surface antigenthat is differentially or preferentially expressed by specific celltypes (e.g., TPC) or by cells under certain conditions (e.g., duringspecific points of the cell life cycle or cells in a particular niche).Preferably, such markers are proteins, and more preferably, possess anepitope for antibodies, aptamers or other binding molecules as known inthe art. However, a marker may consist of any molecule found on thesurface or within a cell including, but not limited to, proteins(peptides and polypeptides), lipids, polysaccharides, nucleic acids andsteroids. Examples of morphological marker characteristics or traitsinclude, but are not limited to, shape, size, and nuclear to cytoplasmicratio. Examples of functional marker characteristics or traits include,but are not limited to, the ability to adhere to particular substrates,ability to incorporate or exclude particular dyes, for example but notlimited to exclusions of lipophilic dyes, ability to migrate underparticular conditions and the ability to differentiate along particularlineages. Markers can also be a protein expressed from a reporter gene,for example a reporter gene expressed by the cell as a result ofintroduction of the nucleic acid sequence encoding the reporter geneinto the cell and its transcription resulting in the production of thereporter protein that can be used as a marker. Such reporter genes thatcan be used as markers are, for example but not limited to fluorescentproteins enzymes, chromomeric proteins, resistance genes and the like.

In a related sense the term marker phenotype in the context of a tissue,cell or cell population (e.g., a stable TPC phenotype) means any markeror combination of markers that may be used to characterize, identify,separate, isolate or enrich a particular cell or cell population (e.g.,by FACS). In specific embodiments, the marker phenotype is a cellsurface phenotype that may be determined by detecting or identifying theexpression of a combination of cell surface markers.

Those skilled in the art will recognize that numerous markers (or theirabsence) have been associated with various populations of cancer stemcells and used to isolate or characterize tumor cell subpopulations. Inthis respect exemplary cancer stem cell markers comprise OCT4, Nanog,STAT3, EPCAM, CD24, CD34, NB84, TrkA, GD2, CD133, CD20, CD56, CD29,B7H3, CD46, transferrin receptor, JAM3, carboxypeptidase M, ADAM9,oncostatin M, Lgr5, Lgr6, CD324, CD325, nestin, Sox1, Bmi-1, eed,easyh1, easyh2, mf2, yy1, smarcA3, smarckA5, smarcD3, smarcE1, mllt3,FZD1, FZD2, FZD3, FZD4, FZD6, FZD7, FZD8, FZD9, FZD10, WNT2, WNT2B,WNT3, WNT5A, WNT10B, WNT16, AXIN1, BCL9, MYC, (TCF4) SLC7A8, IL1RAP,TEM8, TMPRSS4, MUC16, GPRC5B, SLC6A14, SLC4A11, PPAP2C, CAV1, CAV2,PTPN3, EPHA1, EPHA2, SLC1A1, CX3CL1, ADORA2A, MPZL1, FLJ10052, C4.4A,EDG3, RARRES1, TMEPAI, PTS, CEACAM6, NID2, STEAP, ABCA3, CRIM1, IL1R1,OPN3, DAF, MUC1, MCP, CPD, NMA, ADAM9, GJA1, SLC19A2, ABCA1, PCDH7,ADCY9, SLC39A1, NPC1, ENPP1, N33, GPNMB, LY6E, CELSR1, LRP3, C20orf52,TMEPAI, FLVCR, PCDHA10, GPR54, TGFBR3, SEMA4B, PCDHB2, ABCG2, CD166,AFP, BMP-4, β-catenin, CD2, CD3, CD9, CD14, CD31, CD38, CD44, CD45,CD74, CD90, CXCR4, decorin, EGFR, CD105, CD64, CD16, CD16a, CD16b, GLI1,GLI2, CD49b, and CD49f. See, for example, Schulenburg et al., 2010,PMID: 20185329, U.S. Pat. No. 7,632,678 and U.S.P.Ns. 2007/0292414,2008/0175870, 2010/0275280, 2010/0162416 and 2011/0020221 each of whichis incorporated herein by reference. It will be appreciated that anumber of these markers were included in the PhenoPrint Array describedabove.

Similarly, non-limiting examples of cell surface phenotypes associatedwith cancer stem cells of certain tumor types includeCD44^(hi)CD24^(low), ALDH⁺, CD133⁺, CD123⁺, CD34⁺CD38⁻, CD44⁺CD24⁻,CD46^(hi)CD324⁺CD66c⁻, CD133⁺CD34⁺ CD10⁻CD19⁻, CD138⁻CD34⁻CD19⁺,CD133⁺RC2⁺, CD44⁺α₂β₁ ^(hi)CD133⁺, CD44⁺CD24⁺ESA⁺, CD271⁺, ABCB5⁺ aswell as other cancer stem cell surface phenotypes that are known in theart. See, for example, Schulenburg et al., 2010, supra, Visvader et al.,2008, PMID: 18784658 and U.S.P.N. 2008/0138313, each of which isincorporated herein in its entirety by reference. Those skilled in theart will appreciate that marker phenotypes such as those exemplifiedimmediately above may be used in conjunction with standard flowcytometric analysis and cell sorting techniques to characterize,isolate, purify or enrich TIC and/or TPC cells or cell populations forfurther analysis. Of interest with regard to the instant invention CD46,CD324 and, optionally, CD66c are either highly or heterogeneouslyexpressed on the surface of many human colorectal (“CR”), breast (“BR”),non-small cell lung (NSCLC), small cell lung (SCLC), pancreatic (“PA”),melanoma (“Mel”), ovarian (“OV”), and head and neck cancer (“HN”) tumorcells, regardless of whether the tumor specimens being analyzed wereprimary patient tumor specimens or patient-derived NTX tumors.

Cells with negative expression (i.e. “−”) are herein defined as thosecells expressing less than, or equal to, the 95^(th) percentile ofexpression observed with an isotype control antibody in the channel offluorescence in the presence of the complete antibody staining cocktaillabeling for other proteins of interest in additional channels offluorescence emission. Those skilled in the art will appreciate thatthis procedure for defining negative events is referred to as“fluorescence minus one”, or “FMO”, staining. Cells with expressiongreater than the 95^(th) percentile of expression observed with anisotype control antibody using the FMO staining procedure describedabove are herein defined as “positive” (i.e. “+”). As defined hereinthere are various populations of cells broadly defined as “positive.”First, cells with low expression (i.e. “lo”) are generally defined asthose cells with observed expression above the 95^(th) percentiledetermined using FMO staining with an isotype control antibody andwithin one standard deviation of the 95^(th) percentile of expressionobserved with an isotype control antibody using the FMO stainingprocedure described above. Cells with “high” expression (i.e. “hi”) maybe defined as those cells with observed expression above the 95^(th)percentile determined using FMO staining with an isotype controlantibody and greater than one standard deviation above the 95^(th)percentile of expression observed with an isotype control antibody usingthe FMO staining procedure described above. In other embodiments the99^(th) percentile may preferably be used as a demarcation point betweennegative and positive FMO staining and in particularly preferredembodiments the percentile may be greater than 99%.

Using techniques such as those described above to quickly identify andrank colorectal tumor antigens based on expression intensity andheterogeneity across several NTX tumors from colorectal cancer patients,candidate TPC antigens were further assessed by comparison of tumorversus normal adjacent tissue and then selected based, at least in part,on the up- or down-regulation of the particular antigen in malignantcells. Moreover, systematic analysis of a variety of cell surfacemarkers for their ability to enrich for the ability to transplant fullyheterogeneous tumors into mice (i.e. tumorigenic ability), andsubsequent combination of these markers substantially improved theresolution of the method and improved the ability to tailor fluorescenceactivated cell sorting (FACS) techniques to identify and characterizedistinct, highly enriched tumor cell subpopulations that exclusivelycontained all tumor generating ability upon transplantation (i.e. tumorinitiating cells). To reiterate, the term tumor initiating cell (TIC) ortumorigenic (TG) cell encompasses both Tumor Perpetuating Cells (TPC;i.e. cancer stem cells) and highly proliferative Tumor Progenitor cells(TProg), which together generally comprise a unique subpopulation (i.e.0.1-25%) of a bulk tumor or mass; the characteristics of which aredefined above. The majority of tumor cells characterized in this fashionare devoid of this tumor forming ability, and can thus be characterizedas non-tumorigenic (NTG). Surprisingly, it was observed that mostdistinct markers identified using the proprietary PhenoPrint Array didnot demonstrate an ability to enrich tumor initiating cell populationsin colorectal tumors using standard FACS protocols, but that distinctmarker combinations could be used to identify two subpopulations oftumor initiating cells: TPC and TProg. Those skilled in the art willrecognize that the defining difference between TPC and TProg, thoughboth are tumor initiating in primary transplants, is the ability of TPCto perpetually fuel tumor growth upon serial transplantation at low cellnumbers. Furthermore, the marker/proteins used in combination to enrichfor both TPC and TProg were unknown to be associated with cellscontaining such activity in any tissue or neoplasm prior to discovery bycurrent inventors though others have defined cell surface markers orenzymatic activity that can similarly be used to enrich for tumorigeniccells (Dylla et al 2008, supra). As set forth below, specific tumor cellsubpopulations isolated using cell surface marker combinations alludedto above were then analyzed using whole transcriptome next generationsequencing to identify and characterize differentially expressed genes.

Example 2 Isolation and Analysis of RNA Samples from Enriched TumorInitiating Cell Populations

Several established colorectal NTX cell lines (SCRX-CR4, CR11, CR33,PA3, PA6 & PA14) generated and passaged as described in Example 1 wereused to initiate tumors in immune compromised mice. For mice bearingSCRX-CR4, PA3 or PA6 tumors, once the mean tumor burden reached ˜300 mm³the mice were randomized and treated with 15 mg/kg irinotecan, 25 mg/kgGemcitabine, or vehicle control (PBS) twice weekly for a period of atleast twenty days prior to euthanization. Tumors arising from all sixNTX lines, including those from mice undergoing chemotherapeutictreatment were removed and TPC, TProg and NTG cells, respectively, wereisolated from freshly resected colorectal NTX tumors and, similarly, TGand NTG cells were isolated from pancreatic NTX tumors, generally usingthe technique set out in Example 1. More particularly, cell populationswere isolated by FACS and immediately pelleted and lysed in QiagenRLTplus RNA lysis buffer (Qiagen, Inc.). The lysates were then stored at−80° C. until used. Upon thawing, total RNA was extracted using theQiagen RNeasy isolation kit (Qiagen, Inc.) following vendor'sinstructions and quantified on the Nanodrop (Thermo Scientific) and aBioanalyzer 2100 (Agilent Technologies) again using the vendor'sprotocols and recommended instrument settings. The resulting total RNApreparation was suitable for genetic sequencing and analysis.

Total RNA samples obtained from the respective cell populations isolatedas described above from vehicle or chemotherapeutic agent-treated micewere prepared for whole transcriptome sequencing using an AppliedBiosystems SOLiD 3.0 (Sequencing by Oligo Ligation/Detection) nextgeneration sequencing platform (Life Technologies), starting with 5 ngof total RNA per sample. The data generated by the SOLiD platform mappedto 34,609 genes from the human genome and was able to detect ephrin-Aligands, including EFNA4, and provided verifiable measurements of ENFAlevels in most samples.

Generally the SOLiD3 next generation sequencing platform enablesparallel sequencing of clonally-amplified RNA/DNA fragments linked tobeads. Sequencing by ligation with dye-labeled oligonucleotides is thenused to generate 50 base reads of each fragment that exists in thesample with a total of greater than 50 million reads generating a muchmore accurate representation of the mRNA transcript level expression ofproteins in the genome. The SOLiD3 platform is able to capture not onlyexpression, but SNPs, known and unknown alternative splicing events, andpotentially new exon discoveries based solely on the read coverage(reads mapped uniquely to genomic locations). Thus, use of this nextgeneration platform allowed the determination of differences intranscript level expression as well as differences or preferences forspecific splice variants of those expressed mRNA transcripts. Moreover,analysis with the SOLiD3 platform using a modified whole transcriptomeprotocol from Applied Biosystems only required approximately 5 ng ofstarting material pre-amplification. This is significant as extractionof total RNA from sorted cell populations where the TPC subset of cellsis, for example, vastly smaller in number than the NTG or bulk tumorsand thus results in very small quantities of usable starting material.

Duplicate runs of sequencing data from the SOLiD3 platform werenormalized and transformed and fold ratios calculated as is standardindustry practice. As seen in FIG. 2, levels of EFNA1, EFNA3 and EFNA4from a tumor were measured as well as levels of Eph receptors EPHA1,EPHA2 and EPHA10. An analysis of the data showed that EFNA4 wasup-regulated at the transcript level by 1.9-3 fold in the SCRx-CR4 NTXtumor TPC over the NTG population, and 1.2-1.4 fold in TPC over theTProg population, irrespective of whether cells were obtained from micebeing treated with (FIG. 2A) vehicle or (FIG. 2B) 15 mg/kg irinotecan.It will further be appreciated that EFNA1 was also elevated in TPCversus TProg and NTG cells, respectively, although to a lesser extentthan EFNA4. Furthermore, when additional colorectal (SCRx-CR11 & CR33)and pancreatic (SCRx-PA3, PA6 & PA14) tumor samples were analyzed bySOLiD3 whole-transcriptome sequencing, EFNA4 gene expression wassimilarly elevated in TPC versus TProg and NTG cells in colorectalcancer (FIG. 3A) and in the TIC (or TG) subpopulation of cells frompancreatic tumors (FIG. 3B), defined using a panel of unique cellsurface markers discovered as illustrated above (TPC and TProg cellsubsets, which constitute the TIC population in pancreatic tumors havenot yet been defined).

It was also observed that the expression of EPHA2 receptor, with whichboth EFNA4 and EFNA1 ligands interact, inversely reflects that of bothEFNA4 and EFNA1 during the progression of differentiation from TPC toNTG cells. This converse expression pattern of the EFNA1/EFNA4 ligandsand EPHA2 receptor suggests that crosstalk between these ligand/receptorpairs might play a role in cell fate decisions during colorectal cancerstem cell differentiation and that neutralizing these interactions mightnegatively impact tumor growth. Specifically, by blocking EphA2interactions with EFNA1 and/or EFNA4 using neutralizing antibodiesagainst the later pair of ephrin-A ligands, TPC might be sensitized tochemotherapeutic agents, for example, or forced to differentiate.Moreover, by targeting TPC using EFNA1 and/or EFNA4-internalizingantibodies, TPC might be killed directly by the naked modulator orthrough the use of a toxin or antibody drug conjugate.

The observations detailed above show that EFNA1 and/or EFNA4 expressionis generally elevated in TPC populations and suggests that thesemembrane-tethered ligands may play an important role in tumorigenesisand tumor maintenance, thus constituting excellent targets for noveltherapeutic approaches.

Example 3 Real-Time PCR Analysis of Ephrin-A Ligands in Enriched TumorInitiating Cell Populations

To validate the differential ephrin-A ligand expression observed bywhole transcriptome sequencing in TPC populations versus TProg and NTGcells in colorectal cancer, and TG versus NTG cells in pancreaticcancer, TaqMan® quantitative real-time PCR was used to measure geneexpression levels in respective cell populations isolated from variousNTX lines as set forth above. It will be appreciated that such real-timePCR analysis allows for a more direct and rapid measurement of geneexpression levels for discrete targets using primers and probe setsspecific to a particular gene of interest. TaqMan® real-timequantitative PCR was performed on an Applied Biosystems 7900HT Machine(Life Technologies), which was used to measure EFNA4 gene expression inmultiple patient-derived NTX line cell populations and correspondingcontrols. Moreover, the analysis was conducted as specified in theinstructions supplied with the TaqMan System and using commerciallyavailable EFNA4 primer/probe sets (Life Technologies).

As seen in FIG. 4, quantitative real-time PCR interrogation of geneexpression in NTG, TProg and TPC populations isolated from 3 distinctcolorectal NTX tumor lines (SCRx-CR4, CR5 & CR14) showed that EFNA4 geneexpression is elevated more than 1.4-fold in the TIC subpopulations (TPCand/or TProg) versus NTG cells. EFNA4 was also elevated approximately1.8-fold in TIC populations in mice undergoing treatment withirinotecan, and in the TG cell population of pancreatic tumors (e.g.SCRx-PA3). The observation of elevated EFNA4 expression in NTX TIC cellpreparations as compared with NTG cell controls from both colorectal andpancreatic patient-derived NTX tumors using the more widely acceptedmethodology of real-time quantitative PCR confirms the more sensitiveSOLiD3 whole transcriptome sequencing data of the previous Example, andsupports the observed association between EFNA4 and cells underlyingtumorigenesis, resistance to therapy and recurrence.

Example 4 Expression of Ephrin-A Ligands in Unfractionated ColorectalTumor Specimens

In light of the fact that ephrin-A ligand gene expression was found tobe elevated in TPC populations from colorectal tumors when compared withTProg and NTG cells from the same tumors, experiments were conducted todetermine whether elevated ephrin-A ligand (i.e., EFNA4) expression wasalso detectable in unfractionated colorectal tumor samples versus normaladjacent tissue (NAT). Similarly, measurements were also made todetermine how the expression of ephrin-A ligands in tumors compares withlevels in normal tissue samples. Custom TumorScan qPCR (OrigeneTechnologies) 384-well arrays containing 110 colorectal patient tumorspecimens, normal adjacent tissue, and 48 normal tissues were designedand fabricated using art known techniques. Using the procedures detailedin Example 3 and the same EFNA4 specific primer/probe sets, TaqManreal-time quantitative PCR was performed in the wells of the customplates.

FIGS. 5A and 5B show the results of the expression data in a graphicalformat normalized against the mean expression in normal colon and rectumtissue. More specifically, FIG. 5A summarizes data generated using 168tissue specimens, obtained from 110 colorectal cancer patients, (35tissue specimens of which are normal (NL) adjacent tissue fromcolorectal cancer patients) and 48 normal tissues from other locations(Other NL). In the plot, data from each tissue specimen/patient isrepresented by a dot, with the geometric mean value of each populationdemarcated on the X-axis represented as a line. Similarly, FIG. 5Bcontains data from 24 matched colorectal patient specimens obtained fromtumor (T) or normal adjacent tissue (N) at various stages of the disease(I-IV). Here the plotted data is presented on a sample by sample basiswith linkage between the respective tumor and normal adjacent tissuefrom individual patients. Expression of EFNA4 is clearly higher in themajority of matched tumor versus normal adjacent tissue, with thedifferential expression in Stages 2, 3 and 4 reaching statisticalsignificance (n≥4, P≤0.047). Both FIGS. 5A and 5B indicate that, in allfour stages presented, the expressed level of the EFNA4 gene is elevatedin a majority of colorectal tumors and in matched tumor specimens versusnormal adjacent tissue. Moreover, the mean EFNA4 gene expression in anyStage of colorectal cancer appears at least equal to, if not greaterthan, the highest levels of EFNA4 gene expression in any normal tissueinterrogated in these experiments (FIG. 5A). These results demonstratethat EFNA4 expression is increased in colorectal cancer and when coupledwith the above observations that EFNA4 expression is greatest incolorectal TPC and pancreatic TIC, suggests that therapeutic targetingof tumorigenic cells expressing EFNA4 may provide great therapeuticbenefit to cancer patients.

Example 5 Differential Expression of Ephrin-A Ligand in Exemplary TumorSamples

To further assess ephrin-A ligand gene expression in additionalcolorectal cancer patient tumor samples and tumor specimens frompatients diagnosed with 1 of 17 other different solid tumor types,Taqman qRT-PCR was performed using TissueScan™ qPCR (OrigeneTechnologies) 384-well arrays, which were custom fabricated as describedin Example 4. The results of the measurements are presented in FIG. 6and show that gene expression of EFNA4 is significantly elevated orrepressed in a number of tumor samples.

In this regard, FIGS. 6A and 6B show the relative and absolute geneexpression levels, respectively, of human EFNA4 in whole tumor specimens(grey dots) or matched normal adjacent tissue (NAT; white dots) frompatients with one of eighteen different solid tumor types. In FIG. 6A,data is normalized against mean gene expression in NAT for each tumortype analyzed. In FIG. 6B, the absolute expression of EFNA4 was assessedin various tissues/tumors, with the data being plotted as the number ofcycles (Ct) needed to reach exponential amplification by quantitativereal-time PCR. Specimens not amplified were assigned a Ct value of 45,which represents the last cycle of amplification in the experimentalprotocol. Each dot represents an individual tissue specimen, with themean value represented as a black line.

Using the custom Array, it was observed that the majority of patientsdiagnosed with colorectal cancer and most patients diagnosed withendometrial, esophageal, liver, lung, prostate, bladder and uterinecancer had significantly more EFNA4 gene expression in their tumorsversus NAT, suggesting that EFNA4 might play a role in tumorigenesisand/or tumor progression in these tumors. In contrast, expression ofEFNA4 appeared significantly repressed in tumors from patients withadrenal and pancreatic cancer. What was also clear from the thesestudies is that EFNA4 gene expression was generally low to moderate inmost NAT samples; with the highest expression being observed in theadrenal gland, breast, cervix and ovaries. Again, these data suggestthat differential EFNA4 expression (high or low) is indicative, andpotentially dispositive, as to tumorigenesis or perpetuation in patientspresenting selected hyperproliferative disorders.

EFNA4 expression was also assessed using proprietary non-traditionalxenografts (NTX) as discussed above and quantified in relation to normaltissue expression. Quantitative real-time PCR was performed oncommercial normal tissue RNA samples (breast, colon, esophagus, heart,kidney, liver, lung, ovary, pancreas, skeletal muscle, small intestine)and on NTX tumors from breast cancer (BR), colorectal cancer (CR),kidney cancer (KDY), liver cancer (LIV), melanoma (MEL), non-small celllung cancer (NSCLC), ovarian cancer (OV), pancreatic cancer (PA), andsmall cell lung cancer (SCLC). The results, shown in FIG. 6C demonstrateelevated expression of EFNA4 in the breast, colon, and liver NTX linesrelative to expression in normal tissues. Conversely, FIG. 6D documentsexpression of the related family member EFNA1 in many of the same normaland NTX lines and shows little differential expression between normaland tumor tissues. Despite this expression profile EFNA modulators ofthe instant invention that react with EFNA1 (including those that reactwith other EFNA) may effectively be used to eliminate tumorigenic cellsas evidenced in the subsequent Examples.

In any event, to confirm that the elevated mRNA expression detected byquantitative real-time PCR also translates to elevated protein levels ofEFNA4, Western blots were run. Cell lysates of NTX and cell lines (293naive and 293 EFNA4 over-expressing cells) were produced using a totalprotein extraction kit (Bio Chain Institute # K3011010) following theprovided protocol, to match the commercially available normal tissuelysates (Novus Biologicals). Protein concentration of the lysates wasdetermined using a BCA protein assay (Pierce/Thermo Fisher #23225).Equal amounts of cell lysates were run on NuPAGE Novex 4-12% Bis-Trisgels (Life Technologies) in MES Buffer under reducing conditions. Acommercially available antibody against human EFNA4 (R&D Systems—AF369)was used to detect EFNA4 protein expression. In the top panel of FIG.6E, 293 cells engineered to over-express EFNA4 show high expressioncompared to naive 293 cells. Additionally, in the top panel severalbreast, colon and non-small cell lung cancer NTX showed relatively highexpression of EFNA4. Under similar conditions the Western blot in thebottom panel of FIG. 6E shows that normal tissues express low orundetectable levels of EFNA4 when compared with the high EFNA4expression in the NTX cell line CR11. An anti-GAPDH control antibody isused to demonstrate equal loading of cell lysates in both panels.

Example 6 Generation of Anti-EFNA Antibodies Using EFNA Immunogens

EFNA modulators in the form of murine antibodies were produced inaccordance with the teachings herein through inoculation withhEFNA4-ECD-Fc, hEFNA4-ECD-His, hEFNA1-ECD-His, whole cell BALB/c 3T3cells over expressing EFNA4 or the plasma preps prepared as set forthherein (ECD—extracellular domain). Immunogens were all prepared usingcommercially available starting materials (e.g., Recombinant Humanephrin-A4 Fc Chimera, CF R&D systems #369-EA-200) and/or techniques wellknown to those skilled in the art.

More particularly murine antibodies were generated by immunizing 9female mice (3 each: Balb/c, CD-1, FVB) with various preparations ofEFNA4 or EFNA1 antigen. Immunogens included Fc constructs or His taggedhuman EFNA4 or EFNA1, membrane fractions extracted from 10⁷ overexpressing EFNA4 293 cells or whole 3T3 cells over expressing humanEFNA4 on the surface. Mice were immunized via footpad route for allinjections. 10 μg of EFNA4 or EFNA1 immunogen or 1×10⁶ cells or cellequivalents emulsified with an equal volume of TITERMAX™ or alumadjuvant were used for immunization. After immunization mice wereeuthanized, and draining lymph nodes (popliteal and inguinal, ifenlarged) were dissected out and used as a source for antibody producingcells. Lymphocytes were released by mechanical disruption of the lymphnodes using a tissue grinder.

One of two fusion protocols was used. In the first electrofusion with aGenetronic device was performed followed by plating and screening of thepolyclonal hybridomas with a subsequent subcloning to generatemonoclonal hybridomas. In the second ectrofusion with a BTX instrumentwas performed followed by growth of the hybridoma library in bulk andsingle cell deposition of the hybridomas with a subsequent screen of theclones.

Genetronic device fusion protocol: The fusion was performed by mixing asingle cell suspension of B cells with non-secreting P3x63Ag8.653myeloma cells purchased from (ATCC CRL-1580; Kearney et al, J. Immunol.123:1548-1550 (1979)) at a ratio of 1:1. The cell mixture was gentlypelleted by centrifugation at 800 g. After complete removal of thesupernatant, the cells were treated with 2-4 mL of Pronase solution forno more than 2 minutes. Electrofusion was performed using a fusiongenerator, model ECM2001 (Genetronic, Inc.).

Cells were plated at 2×10⁴/well in flat bottom microtiter plates,followed by two weeks incubation in selective HAT medium (Sigma, CRLP-7185). Individual wells were then screened by ELISA and FACS foranti-human EFNA4monoclonal IgG antibodies.

ELISA microtiter plates were coated with purified recombinant EFNA4 Hisfusion proteins from transfected 293 cells at 100 ng/well in carbonatebuffer. Plates incubated at 4° C. overnight than blocked with 200μl/well of 3% BSA in PBS/Tween (0.05%). Supernatant from hybridomaplates were added to each well and incubated for 1-2 hours at ambienttemperature. The plates were washed with PBS/Tween and than incubatedwith Goat anti mouse IgG, Fc Fragment Specific conjugated withhorseradish proxidase (HRP) Jackson ImmunoResearch) for one hour at roomtemperature. After washing, the plates were developed with TMB substrate(Thermo Scientific 34028) and analyzed by spectrophotometer at OD 450.

EFNA4 secreted hybridoma from positive wells were, rescreened andsubcloned by limited dilution or single cell FACS sorting.

Sub cloning was performed on selected antigen-positive wells usinglimited dilution plating. Plates were visually inspected for thepresence of single colony growth and supernatants from single colonywells then screened by antigen-specific ELISAs described above and FACSconfirmation as described below. The resulting clonal populations wereexpanded and cryopreserved in freezing medium (90% FBS, 10% DMSO) andstored in liquid nitrogen. This fusion from mice immunized with EFNA4yielded 159 murine monoclonal antibodies reactive for EFNA4 using theELISA protocol described above.

BTX instrument fusion protocol: A single cell suspension of B cells werefused with non-secreting P3x63Ag8.653 myeloma cells at a ratio of 1:1 byelectrofusion. Electrofusion was performed using the Hybrimune System,model 47-0300, (BTX Harvard Apparatus). Fused cells were resuspended inhybridoma selection medium supplemented with Azaserine (Sigma #A9666)(DMEM (Cellgro cat#15-017-CM) medium containing, 15% Fetal Clone I serum(Hyclone), 10% BM Condimed (Roche Applied Sciences), 1 mM sodiumpyruvate, 4 mM L-glutamine, 100 IU Penicillin-Streptomycin, 50 μM2-mercaptoethanol, and 100 μM hypoxanthine) and then plated in four T225flasks at 90 ml selection medium per flask. The flasks are then placedin a humidified 37° C. incubator containing 5% CO₂ and 95% air for 6-7days.

At 6-7 days of growth the library is plated at 1 cell per well in 48Falcon 96 well U-bottom plates using the Aria I cell sorter. Brieflyculture medium containing 15% Fetal Clone I serum (Hyclone), 10%BM-Condimed (Roche Applied Sciences), 1 mM sodium pyruvate, 4 mML-glutamine, 100 IU Penecillin-Streptamycin, 50 μM 2-mercaptoethanol,and 100 μM hypoxanthine is plated at 200 ul per well in 48 Falcon 96well U-bottom plates. Viable hybridomas are placed at 1 cell per wellusing the Aria I cell sorter and cultured for 10-11 days and thesupernatants are assayed for antibodies reactive by FACS or ELISA forEFNA4 or EFNA1.

Growth positive hybridomas wells secreting mouse immunoglobulins werescreened for murine EFNA4 specificity using an ELISA assay similar tothat described above. Briefly, 96 well plates (VWR, 610744) were coatedwith 1 μg/mL murine EFNA4-His in sodium carbonate buffer overnight at 4°C. The plates were washed and blocked with 2% FCS-PBS for one hour at37° C. and used immediately or kept at 4° C. Undiluted hybridomasupernatants were incubated on the plates for one hour at RT. The platesare washed and probed with HRP labeled goat anti-mouse IgG diluted1:10,000 in 1% BSA-PBS for one hour at RT. The plates are then incubatedwith substrate solution as described above and read at OD 450.

Growth positive hybridomas wells secreting mouse immunoglobulins werealso screened for human EFNA1 specificity using a FACS assay as follows.Briefly 1×10⁵ per well Jurkat cells expressing human EFNA1 wereincubated for 30 minutes with 25-100 ul hybridoma supernatant. Cellswere washed PBS/2% FCS twice and then incubated with 50 ul per sampleDyeLight 649 labeled goat-anti-mouse IgG, Fc fragment specific secondarydiluted 1:200 in PBS/2% FCS. After a 15 minute incubation cells werewashed 2 times with PBS/2% FCS and re-suspended in PBS/2% FCS with DAPIand analyzed by FACS Canto II (BD Biosciences) under standard conditionsand using the HTS attachment. The resulting EFNA1 specific clonalhybridomas were expanded and cryopreserved in CS-10 freezing medium(Biolife Solutions) and stored in liquid nitrogen. This fusion from miceimmunized with EFNA1 yielded 1 hybridoma reactive with EFNA4 asdetermined using FACS analysis. Moreover, FACS analysis confirmed thatpurified antibody from most or all of these hybridomas bind EFNA4 orEFNA1 in a concentration-dependent manner.

Example 7 Sequencing and Humanization of Ephrin-A Ligand Modulators

7(a) Sequencing:

Based on the foregoing, a number of exemplary distinct monoclonalantibodies that bind immobilized human EFNA4 or EFNA1 with apparentlyhigh affinity were selected. As shown in a tabular fashion in FIG. 7Asequence analysis of the DNA encoding mAbs from Example 6 confirmed thatmany had a unique VDJ rearrangements and displayed novel complementaritydetermining regions. Note that the complementarity determining regionsset forth in FIG. 7A (SEQ ID NOS: 8-59 and 70-95) were derived fromVBASE2 analysis.

For initiation of sequencing TRIZOL reagent was purchased fromInvitrogen (Life Technologies). One step RT PCR kit and QIAquick PCRPurification Kit were purchased from Qiagen, Inc. with RNasin were fromPromega. Custom oligonucleotides were purchased from Integrated DNATechnologies.

Hybridoma cells were lysed in TRIZOL reagent for RNA preparation.Between 10⁴ μL and 10⁵ cells were resuspended in 1 ml TRIZOL. Tubes wereshaken vigorously after addition of 200 μl of chloroform. Samples werecentrifuged at 4° C. for 10 minutes. The aqueous phase was transferredto a fresh microfuge tube and an equal volume of isopropanol was added.Tubes were shaken vigorously and allowed to incubate at room temperaturefor 10 minutes. Samples were then centrifuged at 4° C. for 10 minutes.The pellets were washed once with 1 ml of 70% ethanol and dried brieflyat room temperature. The RNA pellets were resuspended with 40 μL ofDEPC-treated water. The quality of the RNA preparations was determinedby fractionating 3 μL in a 1% agarose gel. The RNA was stored in a −80°C. freezer until used.

The variable DNA sequences of the hybridoma amplified with consensusprimer sets specific for murine immunoglobulin heavy chains and kappalight chains were obtained using a mix of variable domain primers. Onestep RT-PCR kit was used to amplify the VH and VK gene segments fromeach RNA sample. The Qiagen One-Step RT-PCR Kit provides a blend ofSensiscript and Omniscript Reverse Transcriptases, HotStarTaq DNAPolymerase, Qiagen OneStep RT-PCR Buffer, a dNTP mix, and Q-Solution, anovel additive that enables efficient amplification of “difficult”(e.g., GC-rich) templates.

Reaction mixtures were prepared that included 3 μL of RNA, 0.5 of 100 μMof either heavy chain or kappa light chain primers 5 μL of 5× RT-PCRbuffer, 1 μL dNTPs, 1 μL of enzyme mix containing reverse transcriptaseand DNA polymerase, and 0.4 μL of ribonuclease inhibitor RNasin (1unit). The reaction mixture contains all of the reagents required forboth reverse transcription and PCR. The thermal cycler program was RTstep 50° C. for 30 minutes 95° C. for 15 minutes followed by 30 cyclesof (95° C. for 30 seconds, 48° C. for 30 seconds, 72° C. for 1.0minutes). There was then a final incubation at 72° C. for 10 minutes.

To prepare the PCR products for direct DNA sequencing, they werepurified using the QIAquick™ PCR Purification Kit according to themanufacturer's protocol. The DNA was eluted from the spin column using50 μL of sterile water and then sequenced directly from both strands.PCR fragments were sequenced directly and DNA sequences were analyzedusing VBASE2 (Retter et al., Nucleic Acid Res. 33; 671-674, 2005).

As briefly alluded to above the genetic arrangements and derived CDRs(from VBASE2 analysis) of several exemplary anti-hEFNA4/hEFNA1antibodies are set forth in a tabular form in FIG. 7A (SEQ ID NOS: 8-59and 70-95). Further, the nucleic and amino acid sequences of these sameexemplary antibody heavy and light chain variable regions are set forthin FIGS. 7B-7N (SEQ ID NOS: 96-147).

7(b) Humanization:

Four of the murine antibodies from Example 6 were humanized usingcomplementarity determining region (CDR) grafting. Human frameworks forheavy and light chains were selected based on sequence and structuresimilarity with respect to functional human germline genes. In thisregard structural similarity was evaluated by comparing the mousecanonical CDR structure to human candidates with the same canonicalstructures as derived from VBASE2 analysis.

More particularly murine antibodies SC4.5, SC4.15, SC4.22 and SC4.47were humanized using a computer-aided CDR-grafting method (AbysisDatabase, UCL Business Plc.) and standard molecular engineeringtechniques to provide hSC4.5, hSC4.15, hSC4.22 and hSC4.47 modulators(Note: the addition of a subsequent numeral following the clone orantibody designation i.e., SC4.47.3 refers to a particular subclone andis not material for the purposes of the instant disclosure unlessotherwise noted or required by context). The human framework regions ofthe variable regions were selected based on their highest sequencehomology to the mouse framework sequence and its canonical structure.For the purposes of the analysis the assignment of amino acids to eachof the CDR domains is in accordance with the Kabat et al. numbering.Several humanized antibody variants were made in order to generate theoptimal humanized antibody with the humanized antibodies generallyretaining the antigen-binding complementarity-determining regions (CDRs)from the mouse hybridoma in association with human framework regions.Humanized SC4.15, SC4.22 and SC4.471 mAbs bind to EFNA4 antigen withsimilar affinity to their murine counterparts while hSC1.5 bound with aslightly lower affinity as measured using the Biacore system.

Molecular engineering procedures were conducted using art-recognizedtechniques. To that end total mRNA was extracted from the hybridomasaccording to the manufacturer's protocol (Trizol® Plus RNA PurificationSystem, Life Technologies). A primer mix comprising thirty-two mousespecific 5′ leader sequence primers, designed to target the completemouse repertoire, was used in combination with 3′ mouse Cγ1 primer toamplify and sequence the variable region of the antibody heavy chains.Similarly thirty-two 5′ Vk leader sequence primer mix designed toamplify each of the Vk mouse families combined with a single reverseprimer specific to the mouse kappa constant region were used to amplifyand sequence the kappa light chain. The V_(H) and V_(L) transcripts wereamplified from 100 ng total RNA using reverse transcriptase polymerasechain reaction (RT-PCR).

A total of eight RT-PCR reactions were run for each hybridoma: four forthe V kappa light chain and four for the V gamma heavy chain (γ1). TheQIAGEN One Step RT-PCR kit was used for amplification, (Qiagen, Inc.).The extracted PCR products were directly sequenced using specific Vregion primers. Nucleotide sequences were analyzed using IMGT toidentify germline V, D and J gene members with the highest sequencehomology. The derived sequences were compared to known germline DNAsequences of the Ig V- and J-regions using V-BASE2 (Retter et al.,supra) and by alignment of V_(H) and V_(L) genes to the mouse germ linedatabase.

From the nucleotide sequence information, data regarding V, D and J genesegments of the heavy and light chains of SC4.5, SC4.15, SC4.22 andSC4.47 were obtained. Based on the sequence data new primer setsspecific to the leader sequence of the Ig V_(H) and V_(K) chain of theantibodies were designed for cloning of the recombinant monoclonalantibody. Subsequently the V-(D)-J sequences were aligned with mouse Iggerm line sequences. Heavy chain genes of SC4.5 were identified asIGHV2-6 (V), and JH3. Analysis of the short CDR3 of the E5 monoclonalantibody heavy chain did not identified a specific mouse D gene. Theheavy chain genes of SC4.15 were identified as IGHV5-6 (V), DSP2.9(D)and JH3. The heavy chain genes of SC4.22 were identified as VHJ558 (V),D segment was identified as DFL16.1e and JH4 (J). The heavy chain genesof SC4.47 were identified as IGHV1-26 (V), P1inv(D) and JH2 (J). Allfour light chains were K class. Light chains genes were identified asIGKV6-15, JK2 for SC4.5 mAb, IGKV6-b and JK5 for SC4.15 mAb, IGKV1-110and JK1 germ line sequence for SC4.22 mAb and IGKV21-7, JK1 germ linesequences for SC4.47 kappa light chain. These results are summarized inTABLE 1 immediately below.

TABLE 2 Mouse Clone Isotype VH DH JH VL JL SC4.5  IgG1/K IGHV2-6 noneJH3 IGKV6-15 JK2 SC4.15 IgG1/K IGHV5-6 DSP2.9 JH3 IGKV6-b JK5 SC4.22IgG2b/K VHJ558 DFL16.1e JH4 IGKV1-110 JK1 SC4.47 IgG1/K IGHV1-26 P1invJH2 IGKV21-7 JK1

The obtained heavy and light chain sequences from all four clones werealigned to the functional human variable region sequences and reviewedfor homology and canonical structure. The result the heavy and lightchain analysis are shown below in TABLES 3 and 4 respectively.

TABLE 3 % Homology % to human homology Human germ line to mouse Clonehuman VH human DH JH sequence sequence SC4.5 VH3-66 IGHD2-21 JH4 82 75SC4.15 VH3-21 IGHD5-5 JH4 88 88 SC4.22 VH1-18 IGHD5-24 JH6 87 83 SC4.47VH1-46 IGHD3-10 JH4 91 76

TABLE 4 % Homology to human germ % Homology to Clone Human VK Human JKline sequence mouse sequence SC4.5 L1 JK2 86 79 SC4.15 A27 JK4 89 76SC4.22 A18b JK1 89 91 SC4.47 L6 JK4 87 84

As the germ line selection and CDR grafting processes appeared toprovide antibodies that generally retained their bindingcharacteristics, there was apparently little need to insert murineresidues in most of the constructs. However, in hSC4.15 the heavy chainresidue 68 was back mutated from Thr (T) to Lys (K) to improve theantibody characteristics.

The amino acid sequences (along with the associated nucleic acidsequence) of the humanized heavy variable region chains and thehumanized kappa light chains for all four antibodies are shown in FIGS.7O-7R (SEQ ID NOS: 148-163) wherein the CDRs in the amino acid sequences(as defined by Kabat et al., supra) are underlined.

More particularly the nucleic acid sequences and corresponding aminoacid sequences of the humanized SC4.5 heavy chain (SEQ ID NOS: 148 and149), and the humanized light chain (SEQ ID NOS: 150 and 151) are shownin FIG. 7O. Similarly, the nucleic acid sequences and correspondingamino acid sequences of the humanized SC4.15 heavy chain (SEQ ID NOS:152 and 153), and the humanized light chain (SEQ ID NOS: 154 and 155)are shown in FIG. 7P. Another embodiment of the invention is illustratedin FIG. 7Q wherein the nucleic acid sequences and corresponding aminoacid sequences of the humanized SC4.22 heavy chain (SEQ ID NOS: 156 and157), and the humanized light chain (SEQ ID NOS: 158 and 159) are shown.In yet another embodiment FIG. 7R shows the nucleic acid sequences andcorresponding amino acid sequences of the humanized SC4.47 heavy chain(SEQ ID NOS: 160 and 161), and the humanized light chain (SEQ ID NOS:162 and 163). As demonstrated in the Examples below each of theaforementioned humanized antibodies functions as an effective EFNAmodulator in accordance with the teachings herein.

In any event the disclosed modulators were expressed and isolated usingart recognized techniques. To that end synthetic humanized variable DNAfragments (Integrated DNA Technologies) of both heavy chains were clonedinto human IgG1 expression vector. The variable light chain fragmentswere cloned into human C-kappa expression vector. Antibodies wereexpressed by co-transfection of the heavy and the light chain into CHOcells.

More particularly, for antibody production directional cloning of themurine and humanized variable gene PCR products into humanimmunoglobulin expression vectors was undertaken. All primers used in Iggene-specific PCRs included restriction sites (AgeI and XhoI for IgH,XmaI and DraIII for IgK, which allowed direct cloning into expressionvectors containing the human IgG1, and IGK constant regions,respectively. In brief, PCR products were purified with Qiaquick PCRpurification kit (Qiagen, Inc.) followed by digestion with AgeI and XhoI(IgH), XmaI and DraIII (IgK), respectively. Digested PCR products werepurified prior to ligation into expression vectors. Ligation reactionswere performed in a total volume of 10 μL with 200 U T4-DNA Ligase (NewEngland Biolabs), 7.5 μL of digested and purified gene-specific PCRproduct and 25 ng linearized vector DNA. Competent E. coli DH10Bbacteria (Life Technologies) were transformed via heat shock at 42° C.with 3 μL ligation product and plated onto ampicillin plates (100μg/mL). The AgeI-EcoRI fragment of the V_(H) region was than insertedinto the same sites of pEE6.4HuIgG1 expression vector while thesynthetic XmaI-DraIII V_(K) insert was cloned into the XmaI-DraIII sitesof the respective pEE12.4Hu-Kappa expression vector.

Cells producing humanized antibodies were generated by transfection ofHEK 293 cells with the appropriate plasmids using 293fectin. In thisrespect plasmid DNA was purified with QIAprep Spin columns (Qiagen).Human embryonic kidney (HEK) 293T (ATCC No CRL-11268) cells werecultured in 150 mm plates (Falcon, Becton Dickinson) under standardconditions in Dulbecco's Modified Eagle's Medium (DMEM) supplementedwith 10% heat inactivated FCS, 100 μg/mL streptomycin, 100 U/mLpenicillin G (all from Life Technologies).

For transient transfections cells were grown to 80% confluency. Equalamounts of IgH and corresponding IgL chain vector DNA (12.5 μg of eachvector DNA) was added to 1.5 mL Opti-MEM mixed with 50 μL HEK 293transfection reagent in 1.5 mL opti-MEM. The mix was incubated for 30min at room temperature and distributed evenly to the culture plate.Supernatants were harvested three days after transfection, replaced by20 mL of fresh DMEM supplemented with 10% FBS and harvested again at day6 after transfection. Culture supernatants were cleared from cell debrisby centrifugation at 800×g for 10 min and stored at 4° C. Recombinantchimeric and humanized antibodies were purified with Protein G beads (GEHealthcare).

Example 8 Characteristics of EFNA Modulators

8(a) General Modulator Characteristics

Various methods were used to analyze the binding characteristics ofselected ephrin-A4 modulators generated as set forth above.Specifically, a number of EFNA4 antibodies were characterized as toaffinity, kinetics, binning, and cross-reactivity with regard tocynomolgus and mouse homologs (generated internally) by ForteBIO®.Western reactivity was also measured and epitopes were determined fortwo antibodies (SC4.22 and SC4.91) that bind under reducing conditions.In addition, the antibodies were tested for their ability to neutralize(i.e. block receptor ligand interaction), internalize and werebenchmarked for their relative EC₅₀ of killing by in vitro cytotoxicityassay using the procedures set forth in these Examples (e.g., seeExamples 12 and 16). The results of this characterization are set forthin tabular form in FIG. 8A.

With regard to the data, affinity was measured in three ways to ensureaccuracy. First, binding signal was measured for a fixed amount ofantibody probed against serial dilutions of antigen in an ELISA todetermine relative modulator activity (data shown for cyno bindingonly). Second, the affinities and kinetic constants k_(on) and k_(off)of the selected modulators were then measured using bio-layerinterferometry analysis on a ForteBIO RED (ForteBIO, Inc.) with astandard antigen concentration series. Finally, the affinity of selectedmodulators was measured by surface plasmon resonance (Biacore System, GEHealthcare). Based on a standard antigen concentration series and usinga 1:1 Langmuir binding model, the K_(d) of the antibody binding toantigen and the kinetic constants k_(on) and k_(off) were determined. Ingeneral, the selected modulators exhibited relatively high affinities inthe nanomolar range.

As to antibody binning, ForteBIO was used per manufacturer'sinstructions to identify antibodies, which bound to the same ordifferent bins. Briefly, an antibody (Ab1) was captured onto ananti-mouse capture chip, a high concentration of nonbinding antibody wasthen used to block the chip and a baseline was collected. Monomeric,recombinant epluin-A4-His was then captured by the specific antibody(Ab1) and the tip was dipped into a well with either the same antibody(Ab1) as a control or into a well with a different antibody (Ab2). Ifadditional binding was observed with a new antibody, then Ab1 and Ab2were determined to be in a different bin. If no further bindingoccurred, similar to the control Ab1, then Ab2 was determined to be inthe same bin. This process can be expanded to screen large libraries ofunique antibodies using a full row of antibodies representing uniquebins in a 96-well plate. This experiment showed the screened antibodiesbound to at least three different bins or epitopes on the EFNA4 protein.

In order to determine whether the epitope recognized by the ephrin-A4modulator comprises contiguous amino acids or is formed by noncontiguousamino acids juxtaposed by secondary structure of the antigen, Westernblots were run under reducing and non-reducing conditions. Moreparticularly, using standard electrophoresis techniques well known inthe art, ephrin-A4 antigen in both states was exposed to the selectedmodulator. As shown in FIG. 8A most ephrin-A4 modulators substantiallyreacted only with antigen where disulphide bonds were intact (NR), whiletwo modulators reacted with both non-reduced and reduced antigen (NR/R).For these antibodies, a Pepspot (JPT) membrane was used to determine thelimits of the antibody recognition by peptide. SC4.22 and SC4.91 werefound to recognize the sequence QRFTPFSLGFE (SEQ ID NO: 164) andRLLRGDAVVE (SEQ ID NO: 165), respectively. Retesting of these peptidesability to bind the peptides of interest by ELISA confirmed that theantibodies were indeed specific to these epitopes.

Finally, cross-reactivity with regard to cynomolgus ephrin-A4 homologswere evaluated in ForteBIO using a concentration series withrecombinantly expressed, monomeric ephrin-A4 antigens. As shown in FIG.8A selected modulators were reactive with the homologs. In particular,SC4.5, SC4.15, SC4.91 and SC4.105 were cross-reactive with mouseephrin-A4, while all antibodies cross-reacted with the highly similarcynomolgus ephrin-A4. ND in the table indicates that the data was notdetermined.

8(b) Humanized Modulator Characteristics

Using techniques set forth above in this Example the humanizedconstructs hSC4.15, hSC4.22 and hSC4.47 were analyzed to determine theirbinding characteristics. Moreover, humanized antibody binding wasdirectly compared with the parent murine antibody for both antibodies toidentify any subtle changes in rate constants brought about by thehumanization process.

More specifically, the affinity of murine SC4.47 was measured by aBiacore using surface plasmon resonance (SPR) to provide the results setforth in FIG. 8B. Based on a concentration series of 25, 12.5, and 6.25nM (generating the curves from top to bottom in the FIGS. 8B and 8C) andusing a 1:1 Langmuir binding model, the K_(d) of the antibody binding toantigen was estimated to be 1.1 nM. Similar experiments then run withthe humanized construct showed equivalent results (FIG. 8C) indicatingthat the humanization process had not adversely impacted the affinity.In this regard the measurements indicated that the humanized constructhad a K_(d) of <1×10⁻¹⁰, which was substantially identical to the parentmurine antibody.

Along with the other techniques set out in this Example, thesemeasurements showed that all humanized ephrin-A4 effectors from Example7 possess desirable qualities. As set out in FIG. 8D, SC4.15 stronglycross-reacts with murine ephrin-A4 homolog thereby facilitatingtoxicology studies. The reactivity of all antibodies for the cynomolgusantigen by ELISA could not be distinguished from human EFNA and so isexpected to be very similar.

Example 9 Ephrin-A Ligand Modulators Demonstrate Cell Surface Binding

Supernatants from hybridomas producing antibodies raised againsthEFNA4-Fc as set forth above were screened for cell surface binding asmeasured in a flow cytometric assay. To demonstrate the bindingproperties of the antibodies two cell lines, JurkatE6 cells and Z138cells each of which are known to express high levels of surfaceephrin-A4, were employed. More specifically six million Jurkat E6 cells,stained with the cell labeling dye CFSE (for simple identification), andfour million unlabeled Z138 cells, incubated with 20 μg/ml Fc blockingreagent (Trueblock, Biolegend, Inc.) were mixed to a final concentrationof 1 million cells/mL. 50 μL of this cell mixture was added to 50 μL ofantibody-containing supernatant in each well and incubated for 60minutes at 4° C. The cells were washed once with PBS containing 2% FBS,2 mM EDTA and 0.05% sodium azide (wash buffer) and then stained for 60minutes at 4° C. in the dark with a Fe-region specific F(ab)2 fragmentof Goat-anti-mouse IgG polyclonal antibody conjugated to DyLight649(Jackson Immuno Research). Cells were washed twice with wash buffer, andcounterstained with 2 μg/ml DAPI. Negative control samples were a MouseIgG1 isotype antibody (10 μg/ml, Biolegend, Inc.) and supernatant from ahybridoma (H13.2) known to not secrete mouse IgG. Positive controlsamples were prepared using 10 μg/ml of a purified antibody (SC4.76.2aka E76.2) identified before by ELISA to be EFNA4 specific (left side ofFIG. 9). Samples were collected on a FACS Canto II (BD Biosciences)under standard conditions and using the HTS attachment. Eighty four (84)clones of one hundred fourteen (114) were judged to display significantcell surface binding as demonstrated by flow cytometry through thestaining of both cell lines significantly above negative controlsamples. In this regard FIG. 9 shows the relative binding capacity offifty exemplary hybridoma supernatants.

Example 10 Selected EFNA4 Modulators Neutralize Ephrin-A4 Ligand Binding

Supernatants from hybridomas producing antibodies known to bind to ENFA4expressing cells (Example 9) were tested for their ability to block thebinding of soluble hEFNA4-Fc to bind its receptors (EphAs) on thesurface of HEK293Td cells. Initially, as seen in FIG. 10A HEK293Td cellsare shown to bind hEFNA4-Fc in a dose-dependent manner when compared toa negative control antibody. To demonstrate neutralization of thisbinding 60 μl of anti-EFNA4 hybridoma supernatants were incubated with200 ng/ml hEFNA4-Fc diluted in wash buffer for 2 hours at 4° C. Themixture was then added to fifty thousand HEK293Td cells and incubatedfor 1 hour at 4° C. Cells were washed once in wash buffer and thenstained for 45 minutes at 4° C. in the dark with Fc-region specificF(ab)2 fragment of goat-anti-mouse IgG polyclonal antibody conjugated toDyLight649 (Jackson Immuno Research). Cells were then washed twice withwash buffer, and counterstained with 2 μg/ml DAPI. Negative controlsamples were unstained cells, cells stained with supernatants from anon-IgG producing hybridoma (H13.2) and cells stained with a human IgGFcγ1 fragment. Positive control samples were hEFNA4-Fc stained cells inabsence of hybridoma supernatants and hEFNA4-Fc stained samples inpresence of non-IgG producing hybridoma supernatant (left side of FIG.10B). Samples were measured on a FACS Canto II as previously discussed.As evidenced by FIG. 10B sixty two (62) clones of eighty three (83)tested demonstrated some ability to neutralize hEFNA4-Fc binding to itscell surface receptors when measured using flow cytometry.

Example 11 EFNA Modulators Block Cell Surface EFNA Binding in aConcentration Dependent Manner

To further measure the ability of the ephrin-A ligand modulators of theinstant invention to neutralize EFNA activity, anti-EFNA4 antibodiesfrom selected hybridomas were purified and used as sterile reagents inPBS buffer. Initially a full dose response curve of human and murineEFNA4-Fc (Recombinant Murine ephrin-A4 Fc Chimera, CF R&D Systems) alonewas set up in parallel to demonstrate dose-limited binding of EFNA4-Fcto HEK293Td cells (FIG. 11A). Once this control had been established,serial dilutions of anti-EFNA4 antibodies obtained from three exemplaryhybridomas (i.e. SC4.15.3, SC4.47.3 and SC4.76.2) were incubated withlimiting concentrations (0.1 μg/ml and 1.0 μg/ml) of hEFNA4-Fc andmEFNA4-Fc respectively in wash buffer for 1 hr at 4° C. The resultingreagent mixtures were then transferred to fifty thousand HEK293Td cellsand incubated for 1 hour at 4° C. Cells were washed once in wash bufferand then stained for 45 minutes at 4° C. in the dark with a Fc-regionspecific F(ab)2 fragment of goat-anti-mouse IgG polyclonal antibodyconjugated to DyLight649 (Jackson Immuno Research). Cells were washedtwice with wash buffer, and counterstained with 2 μg/ml DAPI. Negativecontrol samples were unstained cells and cells stained with a human IgGFcγ1 fragment. Samples were collected on a FACS Canto II as previouslyalluded to above. FIG. 11B shows the activity of mAb SC4.15.3 whichpartially inhibits human and mouse EFNA4-Fc binding to cells atrelatively high concentrations. FIG. 11C illustrates the activity of mAbSC4.47.3 which almost completely blocks the ability of hEFNA4-Fc to bindto cells but not the ability of mEFNA4-Fc. Similarly, FIG. 11Ddemonstrates the ability of ephrin-A ligand modulator mAb SC4.76.2 tosubstantially inhibit the ability of hEFNA4-Fc to bind to cells whilenot dramatically impacting the ability of mEFNA4-Fc to bind to thecells. These results are strongly indicative of the capacity of selectedmodulators of the instant invention to inhibit the binding of ephrin-Aligands to cell surface receptors and thus inhibit any associatedtumorigenic activity.

Example 12 EFNA Modulators Block Binding of EFNA to EphA Receptors in aConcentration Dependent Manner

As discussed above EphA2 is a known binding partner for EFNA4. Toexploit this known relationship the extracellular domain of EphA2 wasfused to the Fc portion of a human IgG using standard techniques,transiently expressed in HEK293Td cells and purified from thesupernatant of the culture using Protein A affinity chromatography. Asseen in FIG. 12A the EphA2-Fc homodimer binds in a dose dependent tofashion to Jurkat cells (known to express EFNA) while the Fc portion ofhuman IgG alone does not show any binding. This binding of EphA2-Fc toJurkat cells can be inhibited using the ephrin-A modulators of theinstant invention and, in particular, through the use of monoclonalantibodies to ephrin-A4. To this end fifty thousand Jurkat cells perwell were incubated with 10 μg/ml of four selected anti-ENFA4 antibodies(i.e. SC4.22, SC4.31.3, SC4.47.3 and SC4.73, all prepared as describedabove) in wash buffer for 1 hr at 4° C. Mouse IgG and no antibody (datanot shown) serve as negative controls. After washing, serial dilutionsof EphA2-Fc were added to the cells in wash buffer for 1 hr at 4° C. toprovide the results graphically represented in FIG. 12B. A review ofFIG. 12B shows that modulators SC4.31.3 and SC4.47.3 substantiallyinhibit the binding of EphA2-Fc to EFNA4 whereas modulators SC4.22 andSC4.73 exhibit relatively less inhibition. To further illustrate theability of the disclosed modulators to inhibit interactions with thereceptor, Jurkat cells were first incubated with serial dilutions ofantibodies, followed by incubation with 10 μg/ml EphA2-Fc. The cellswere then washed twice with wash buffer, counterstained with 2 μg/mlDAPI, and analyzed on a FACS Canto II (BD Biosciences) under standardconditions using the HTS attachment to provide the data represented inFIG. 12C. As with FIG. 12B, FIG. 12C demonstrates that modulator mAbSC4.47.3 is a relatively potent inhibitor and efficiently blocks thebinding of EphA2-Fc to EFNA4 expressed on Jurkat cells. By way ofcomparison the other modulators show somewhat less activity withSC4.31.3 providing a moderate amount of inhibition at higherconcentrations.

To extend these findings, interactions between additional EFNA4modulators and EphA receptors were explored. Experiments were carriedout similar the one described above except that HEK293T cellsoverexpressing EFNA4 by means of retroviral transduction (referred to asHEK293T.hEFNA4 cells) (FIG. 12D) or HEK293T cells overexpressing EFNA1by means of retroviral transduction (FIG. 12E) were used. In addition,the assay was carried out at a single EphAx-Fc concentration (10 μg/ml).The data show that SC4.2, SC4.31, and SC4.47 are able to block bindingof all tested EphA receptor binding partners to ephrin-A4 ligand (i.e.,EphA2, EphA3, EphA4, EphA6, EphA7, EphA8 and EphA10. In addition it wasestablished that EFNA4 modulator SC9.65, which was generated in animmunization campaign against EFNA1, (as per Example 6) has the abilityto interfere with the binding of EphA1, EphA2, EphA4 and EphA7 toephrin-A1 ligand. These data, when combined with the results of theother Examples herein, suggest that this modulator ability to antagonizethe binding of various receptors may be significant in providing theobserved therapeutic effects of the instant invention.

Example 13 Modulators to Human Ephrin-A Cross-React with the MouseOrtholog

In light of the fact that the extracellular domains of human and mouseephrin-A4 ligand share 80% sequence identity on the protein level, thedisclosed modulators to human EFNA4 were tested to see if theyassociated with the mouse homolog. More specifically, an antibodysandwich ELISA was used to determine the level of cross-reactivity ofhEFNA4 specific monoclonal antibodies with its mouse homolog. A highprotein binding 96-well assay plate was coated with 0.5 μg/ml of adonkey-anti-human IgG polyclonal antibody specific for the Fc portion ofthe IgG molecule. The protein coating of the plate occurred in 100 μlvolume per well using a 50 mM Sodium Carbonate buffer (pH9.6) during a16 hour incubation at 4° C. Human and mouse EFNA4 molecules fused to theFcγ1 potion of a human IgG molecule (EFNA4-Fc) were serially diluted inPBS buffer containing 2% (w/v) bovine serum albumin (PBSA). Afterwashing the coated plate in PBS buffer containing 0.05% Tween20 (PBST),100 μl per well diluted mouse or human EFNA4-Fc in PBSA was added towells for the duration 3 hours at ambient temperature. The plate wasthen washed again with PBST and 100 μl/well PBSA containing 10% spenthybridoma supernatant or 1 μg/ml purified monoclonal antibody (aspositive control) was added to the plate for the duration of 1 hour atambient temperature. After washing the plate with PBST, 100 μl per wellof PBSA containing a 1:5000 dilution of goat anti-mouse IgG polyclonalantibody, specific for the Fc portion of Mouse IgG and conjugated tohorseradish peroxidase (Jackson Immuno Research), was added to the platefor 30 minutes at ambient temperature. After washing the plateextensively with PBST, 100 μl per well TMB substrate (Thermo Fisher) wasadded to the wells for 15 minutes. The enzymatic reaction was stopped byadding 100 μl/well 2M sulfuric acid. The absorbance of this colorimetricassay was measured at 450 nm using a Victor plate reader (Perkin Elmer).Data are presented as mean absorbance reading plus standard deviationusing two replicates. FIG. 13A shows an exemplary monoclonal antibodySC4.31.3 that recognizes hEFNA4 but not mEFNA4. Conversely FIG. 13Bshows the binding of exemplary monoclonal antibody SC4.91.4 whichrecognizes both human and mouse EFNA4.

To confirm these results the assay was run using the humanized ephrin-A4modulator hSC4.15. More particularly titrated amounts of human and mouseephrin-A4-His were coated on high protein binding 96-well plates in PBSat 4° C. for 16 hours. After blocking the plates for 2 hr at ambienttemperature in PBSA, 0.5 μg/ml of hSC4.15 modulator was added for 2hours in PBSA. The ELISA was developed as described above using adonkey-anti human IgG polyclonal antibody conjugated to horseradishperoxidase (Jackson Immuno Research). FIG. 13C shows that the hSC4.15modulator recognizes both human and mouse ephrin-A4 ligand equally wellindicating that the disclosed humanized modulators are entirelycompatible with the teachings herein.

Example 14 Ephrin-A Ligand Expression in Exemplary Tumor Samples, TumorCell Subpopulations and Hematopoietic Cells

After documenting elevated gene expression levels and generatingantibodies against EFNA4 in the previous Examples, evidence was soughtfor corresponding EFNA4 protein expression is selected cell populations.In this respect, reverse phase cancer protein lysate arrays (ProteoScanArrays; OriGene Technologies) comprising 4 dilutions of 432 tissuelysates from 11 tumor types, or their respective normal adjacent tissue,were provided along with controls consisting of HEK 293 cells without orwith TP53-overexpression driven by an exogenous promoter. EFNA4 proteinexpression in the lysates on this array were detected using a mousemonoclonal EFNA4 antibody of the instant invention that recognizes EFNA4protein by Western Blot (e.g. clone E47.3 aka SC4.47.3). Colorimetricdetection reagents and protocols were provided by the manufacturer ofthe ProteoScan Arrays, spots on the fabricated array were converted to adigital image using a flatbed scanner using BZScan2 Java Software(INSERM-TAGC) to quantify spot intensity.

Selected results of such assays are shown in FIG. 14, and indicate thatexpression of the EFNA4 protein is upregulated in colorectal tumorsamples. More specifically, FIG. 14A shows that EFNA4 protein expressionappears significantly elevated in a subset of colorectal tumorspecimens; especially in patients with Stage IV disease when compared tonormal adjacent tissue or tumor tissue from specimens obtained fromearlier stages of disease. Data was generated as described above andrepresented as average pixel intensity per spot (spot intensity). Thehorizontal black bar in each sample represents the mean for specimens ineach respective category.

After confirming that EFNA4 protein was upregulated in certaincolorectal whole tumor cell lysates tests were conducted to establishthat the same target was expressed on tumor initiating cells. Morespecifically, to determine whether EFNA4 protein expression could bedetected on the cell surface of tumor initiating cells, tumors weredisassociated as described above for flow cytometric analysis. After thetumor sample (e.g. colorectal cell line CR33 as per Example 2) wasdisassociated to a single cell suspension, they were incubated at 37° C.for 24 hours to facilitate antigen re-expression (due to the enzymaticsensitivity of the EFNA4 antigen to collagenase/hyaluronidase), and thenstained with a phycoerythrin (PE)-conjugated monoclonal antibody able torecognize EFNA4. The cells were then analyzed as in previous exampleswith a FACS Canto II (BD Biosciences) under standard conditions usingthe HTS attachment. In conducting such experiments it was observed thatEFNA4 expression was noticeably higher on the TIC cell subpopulation (asdefined by co-staining of the cells with antibodies recognizingTIC-defining cell surface markers; e.g. 46⁺, 324⁺, 66⁻) than on NTGcells. A representative result from an experiment using SCRx-CR33colorectal NTX tumor cells and EFNA4 modulator SC4.47.3 shows that theexpression of EFNA4 was more than 2-fold higher on TIC than on NTG cells(FIG. 14B).

To further confirm that EFNA4 is relatively highly expressed on TICcells, LU86 and LU64 cells were cultured in vitro for 10 days andexpression was measured by flow cytometry using a PE conjugated SC4.47antibody as set forth herein. Resulting colonies were harvested andstained as described above. As illustrated in FIG. 14D the TICpopulation of LU86 cells (solid black line) express EFNA4 well aboveisotype control (shaded gray) and the NTG population (dashed black line)from the same tumor line. Additionally, LU86 cells cultured in vivo canbe killed with EFNA4 antibodies (as shown in Example 16 below).Conversely LU64 cells were found not express elevated levels of EFNA4(FIG. 14D) and subsequently were not killed with anti-EFNA antibodies.

While it is believed that EFNA4 protein expression has not been assessedin solid tumor specimens prior to the instant disclosure, it has beenreported that the protein is expressed at relatively low levels onB-cells and elevated on B-cells from Chronic Lymphocytic Leukemia (CLL)patients. In order to confirm the expression of EFNA4 proteins on normalperipheral blood mononuclear cells (PBMC) assays were run as previouslydescribed in this Example to provide the data set forth in FIG. 14C. Areview of the plots presented in FIG. 14C shows that when EFNA4expression was gauged on PBMC from a normal donor, only CD19⁺ B-cellswere weakly positive, confirming reports in the literature as to whereEFNA4 is expressed.

These data support the observations in above examples that EFNA4overexpression is associated with TIC and/or TPC in colorectal cancer,and may be involved in proliferation and/or survival. The data furthershows that EFNA4 is not expressed on the majority of normal PBMC, andthat expression on normal B-cells is minimal. In view of the forgoingExamples showing: a) EFNA4 gene expression is associated with the TPCcell subpopulation in colorectal cancer and the tumorigenic cellsubpopulation in pancreatic tumors; b) that EFNA4 protein expression ishigher on the TIC cell subpopulation; c) EFNA4 protein expression iselevated in whole tumor specimens from late stage colorectal cancer; andd) the general observation is that TIC are more frequent in late stagetumors, it appears that EFNA4 is associated with those cells underlyingtumor growth, resistance to therapy and tumor recurrence, advocatingthat EFNA4 may play in integral role in supporting TPC and/or TIC in theabove mentioned tumors.

Example 15 Ephrin-A Ligand Modulators are Internalized by K562 Cells

Given the expression profile of ephrin-A ligands established in theprevious Examples, assays were conducted to see if the modulators of theinstant invention were internalized upon binding to the cell surfaceantigen. In this respect supernatant from hybridomas producingantibodies raised against EFNA4-Fc in Example were screened for theirability to internalize in K562 cells, which express EFNA4 at low levelson the cell surface. K562 cells at a starting concentration of 10⁶/ml(single cells suspension) were blocked with Human TruStain (BioLegend,Inc.) for 10 minutes at room temperature, and then diluted to 5×10⁴cells per well. Duplicate samples were then stained for 30 minutes onice with anti-EFNA antibody containing supernatant for a final volume of50 ul. Cells were then washed with FACS staining medium (FSM; 2% fetalbovine serum/Hank's buffered saline solution/25 mM HEPES [pH7.4]) toremove unbound antibody. This was followed by a second stain with donkeyanti-mouse Alexa647 (Life Technologies) for 30 minutes on ice. Sampleswere washed again to remove unbound antibody and then re-suspended ininternalization medium (2% fetal bovine serum/Iscove's ModifiedDulbecco's Medium). To allow internalization, samples were incubated in5% CO2 @ 37° C. (or 4° C. for the Control) for an hour. Internalizationwas stopped by transferring samples to ice and adding excess ice coldFSM. To remove any antibody that did not internalize and remained on thecell surface, samples were treated with low pH phosphate buffered saline(PBS [pH2.0]) for 10 minutes on ice. Following this “acid strip”procedure, samples were washed extensively with FSM, re-suspended in 150μl of FSM containing 2 μg/ml of DAPI, and analyzed by flow cytometry(again using a FACS Canto II (BD Biosciences) under standard conditionsusing the HTS attachment). Any signal detected beyond background resultsfrom antibody internalization: a process that protects the fluorescentconjugate from removal from the cell surface during the acid stripprocess. All incubations were performed in FSM unless stated otherwise.

Screening of 159 EFNA4 antibody-containing hybridoma supernatant clonesusing the acid strip protocol described above showed many supernatantsdisplay a positive shift in fluorescence vs. the IgG negative controlantibodies (data not shown). The exemplary SC4.5, SC4.22 and SC4.73clones, for instance, demonstrated internalization in as far assupernatants from these clones was able to internalize and protect theAlexa647 secondary antibody from acid stripping (FIG. 15A). Compared tothe IgG controls, approximately 15% of the EFNA4 antibody-containingsupernatants induced internalization to varying degrees, with the topnineteen (19) demonstrating a Delta Mean Fluorescent Intensity (MFI at37° C. vs. 4° C.) above 150 (FIG. 15B). This data demonstrates that asubset of modulators generated against human EFNA4 ECD bind the antigenas it is presented on cells and is able to internalize. Such resultsunderscore the potential therapeutic value of ephrin-A ligands astargets for the modulators of the instant invention with or withoutcytotoxic payloads.

The assay was repeated using selected purified EFNA4 modulators at aconcentration of 10 μg/ml and HEK293T (FIG. 15C) and HEK293T.hEFNA4(FIG. 15D) cells as target cells. Parental HEK293T express a low levelof ephrin-A4 ligand on their cell surface. Following the protocoldescribed above, the data demonstrate that all tested ephrin-A4modulators are internalized upon binding to ephrin-A4 ligand expressedon the surface of cells. Recorded mean fluorescence intensities (MFI)for each sample were compared against a standard bead (Becton DickensonSpherotech 8-color rainbow beads) containing eight different knownamounts of encapsulated fluorophore (data not shown). This permitted thetransformation of MFI values into linear values and calculation ofrelative receptor number per cell.

Example 16 EFNA4 Modulators as Targeting Moieties

Targeting of a cytotoxic drug stably linked to an antibody represent anempowered antibody approach that might have great therapeutic benefitfor patients with solid tumors. To determine whether the EFNA4-specificantibodies described above were able to mediate the delivery of acytotoxic agent to live cells, an in vitro cell killing assay wasperformed wherein streptavidin conjugated to the ribosome-inactivatingprotein Saporin (Advanced Targeting Systems) was bound to biotinylatedEFNA4 antibodies, and the ability of these Saporin complexes tointernalize and kill cells was measured 72 hours later by measuring cellviability.

Specifically, 10⁵ Z138 cells per well were plated in wells of a 96-wellplate. The anti-EFNA4 antibodies described above were purified fromsupernatants, biotinylated and then diluted to 20 μg/mL. The Z138 cellline (ATCC CRL-3001) was derived from a patient with mantle celllymphoma and expresses modest amounts of EFNA4. An aliquot of eachantibody, respectively, was mixed 1:1 with streptavidin-ZAP (AdvancedTargeting Systems), vortexed for 5 seconds, and then incubated at roomtemperature for 1 hour. Two additional serial 10-fold dilutions of theantibody-Saporin complexes were then made 50 μL of each mixture,respectively, was added to Z138 cell containing wells. Thecell/antibody-saporin mixture was then incubated at 37° C./5% CO₂ for 24hours. Following this incubation, cells were spun down in theround-bottom 96-well plates, supernatant was removed, and 100 μL offresh culture medium was added to each well. The cells were thenincubated for an additional 72 hours and then viable cell numbers wereenumerated using CellTiter-Glo (Promega Corp.) per the manufacturer'sprotocol.

Using this protocol several antibodies that were able to internalize asdescribed in the previous Example were also able to mediate cell killingin vitro (data not shown) whereas a biotinylated isotype controlantibody was not able to kill cells. That is, several of theseinternalizing modulators were able to mediate Saporin toxininternalization that resulted in cell death. FIG. 16A illustrates thiscell killing capacity for the exemplary internalizing modulator SC4.5.3where the downward slope of the curve represents cell death in aconcentration dependent manner as compared to the control. These dataclearly demonstrate the effectiveness of the disclosed modulators whenacting as vectors for the selective internalization of cytotoxicpayloads in tumorigenic cells expressing ephrin-A ligands.

To corroborate the these results and determine whether EFNA4 effectorscan mediate toxin internalization and cell killing of primary humantumor cells, mouse lineage-depleted NTX cells (i.e. human tumor cellspropagated as low-passage xenografts in immunocompromised mice) wereplated and subsequently exposed to anti-EFNA4 antibodies and Fab-ZAP.

Specifically, NTX tumors representing lung and skin tumor specimens weredissociated into a single cell suspension and plated on BD Primaria™plates (BD Biosciences) in growth factor supplemented serum free mediaas is known in the art. After 3-5 days of culture at 37° C./5% CO₂/5%O₂, cells were contacted with a control (IgG1 or IgG2b) or a murineEFNA4 modulator (SC4.5, SC4.22, SC4.47, or SC4.91 at 1 nM;), and Fab-ZAP(at 40 nM). Modulator-mediated saporin cytotoxicity was then assessed byquantifying the remaining number of cells using CellTiter Glo 5-7 dayslater. As seen in FIG. 16B exposure to the EFNA4 antibodies resulted inreduced LU86 cell numbers, whereas the IgG2b and IgG1 isotype controlantibody did not impact the number of live cells after treatment. InFIG. 16C exposure to SC4.5, SC4.47, SC4.91 antibodies resulted inreduced SK19 cell numbers whereas isotype controls and SC4.22 wereineffective. Not only does this data demonstrate that exemplaryantibodies described herein are specific to EFNA4, are able to bindEFNA4 antigen on the cell surface and facilitate the delivery of acytotoxic payload resulting in cell death, but the above data alsodemonstrated that multiple anti-EFNA4 antibodies can mediate killing ofmultiple NTX tumor cells.

In a variation of the aforementioned killing assay, delivery of acytotoxic payload via EFNA modulators was demonstrated for additionalantibodies and in additional cells. 2000 cells/well of the followingcell types were plated into 96 well tissue culture plates in theirrespective culture media one day before the addition of antibodies andtoxin: HEK293T cells (FIG. 16C), HEK293T.hEFNA4 cells (FIG. 16D).Purified (‘naked’) mouse monoclonal antibodies at various concentrationsand a fixed concentration of 10 nM Anti-Mouse IgG Fab fragmentcovalently linked to Saporin (Advanced Targeting Systems, #IT-48) wereadded to the cultures for 72 hr. Viable cell numbers were enumerated asdescribed above. Raw luminescence counts using cultures containing cellswith the Saporin Fab fragment were set as 100% reference values and allother counts calculated accordingly (referred to as “Normalized RLU”).

Using this assay, we are able to demonstrate that all tested EFNAantibodies but not isotype control antibodies are able to kill targetcells. This assay further demonstrates that internalization occurssolely because of binding of the EFNA4 antibody to the cell surfacewithout the need for additional cross-linking. Finally, the datademonstrate that only cells that express a sufficient number of EFNA ontheir surface are killed by EFNA modulators. Parental HEK293T cellsexpress a low number of EFNA on their cell surface while HEK293T.hEFNA4cells express this ligand strongly (see FIGS. 15C and 15D from theprevious Example). Table 5 below lists half-maximal effectiveconcentration (commonly referred to as “EC50) for all testedantibody/target cell combination. In addition to the aforementioned celllines PC3 cells (ATCC CRL-1435), a cell line derived from a humanadenocarcinoma, was used as a target cell.

TABLE 5 EFNA Modulators Deliver a Cytotoxic Payload EC50 (pM) HEK293THEK293T.hEFNA4 PC3 Z138 Isotype No killing No killing No killing Nokilling SC4.2.1 No killing 10.1 N.T. N.T. SC4.5.1 No killing 15.0 N.T. 4.6 hSC4.15 N.T. 13.7 5.4 N.T SC4.22.1 No killing 28.6 5.4 18.7SC4.31.3 No killing 14.2 N.T. 33.8 SC4.47.3 201 23.2 2.5  9.6 SC4.91.4No killing 7.8 N.T. 15.8 SC4.105.4 No killing 17.3 N.T. 65.2 SC9.65 Nokilling 28.9 N.T. N.T. (N.T. = not tested)

In another variation of the in vitro killing assay, humanized EFNAmodulators were tested for their ability to internalize and deliver acytotoxic payload. The assay was carried out just as described above,except that only 500 cells/well were plated and Anti-Human IgG Fabfragment covalently linked to saporin (Advanced Targeting Systems,#IT-51) was added to the cultures. FIG. 16E illustrates that humanized(Hz in FIG. 16E) EFNA modulators described in Example 7 are able to bindto ephrin-A4 ligand expressed on the surface of target cells and induceinternalization of EFNA together with bound antibody and cytotoxicpayload.

In yet another variation of the in vitro killing assay, humanized EFNAmodulator hSC4.15 shown to bind to mouse and human EFNA equally well(see FIG. 13C) was tested for its ability to internalize and deliver acytotoxic payload to HEK293T cells overexpressing human or mouse EFNA.To ensure direct comparability, lentivirally transduced cells werestained with hSC4.15 and sorted by FACS for moderate expression ofeither human or mouse ephrin-A4 (data not shown). The killing assay wascarried out just as described above. FIG. 16F illustrates that humanizedSC4.15 modulator kills cells expressing mouse or human EFNA equallywell.

Example 17 EFNA Modulators Detect Secreted Ephrin-A Ligand

As discussed in some detail above EFNA4 can exist as GPI-linked moleculeassociated with cell membranes or as secreted truncated ligands orisoforms. Detection of these secreted compounds in biological material,such as bodily fluids or cell culture media, may be useful fordiagnostic purposes or as an aid in patient management (utility asbiomarker). For example it has been suggested that that secreted EFNA4may be found at elevated concentrations in B cell chronic lymphocyticleukemia (B-CLL) patients (Alonso-C L M et al., 2009, Leukemia Research33: 395-406). In order to demonstrate such preferred aspects of theinstant invention, disclosed modulators were used recognizenon-overlapping epitopes of purified EFNA4 and generally detect andquantify secreted EFNA ligands in selected tumorigenic samples. Withregard to this latter feature of the instant invention EFNA modulatorswere used to detect and quantify secreted ephrin-A ligand in human serum(data not shown) and human plasma obtained from B-CLL patients and fromsera of mice bearing human tumor xenotransplants (e.g. as described inExample 1 above). In each case the modulators were able to effectivelymeasure ligand levels as described immediately below.

To detect soluble human EFNA4, antibody SC4.91 was absorbed to ahigh-protein binding microtiter plate, (Greiner BioOne Microlon plates),at 5 μg/ml in 50 mM sodium carbonate buffer (pH9.6) during an overnightincubation at 4° C. After washing the plate in phosphate buffered saline(PBS) containing 0.05% (v/v) Tween20 (PBST), the plate was blocked inPBS containing 2% (w/v) bovine serum albumin (PBSA) for 2 hours atambient temperature. Purified ephrin-A4-His, expressed transiently inCHO-S cells and purified using sequentially Nickel NTA resin and gelfiltration, was serially diluted in PBSA and added for 2 hours to theplate. After washing with PBST, biotinylated antibody SC4.47 was addedat 1 μg/ml in PBSA for 1 hour to the plate. The plate was then washedwith PBST and then Streptavidin-horseradish peroxidase conjugate (e.g.Jackson Immuno Research) was added at a 1:5000 dilution into PBSA for 30minutes. The treated plate was then washed again in PBST and TMBsubstrate solution (e.g. Thermo Fisher) was added for 30 minutes. Thecolor reaction was stopped by adding an equal volume of 2M sulfuric acidafter which the plate was read using an absorbance reading of 450 nm ina standard plate reader. Results of the experiments are shown in FIGS.17A-C.

Using the techniques described immediately above the concentration ofsoluble hephrin-A4-His was plotted against absorbance values to providethe curves shown in FIG. 17A. More specifically, the primary curve showsresults of absorbance measurements at soluble EFNA4 concentrations from0-40 pg/ml while the inset shows the same curve at concentrations from0-1,000 pg/ml. Those skilled in the art will appreciate that thestandard curves shown in FIG. 17A may be used to provide an extremelysensitive assay for the measurement of EFNA4 concentrations inbiological samples.

Taking advantage of the aforementioned measurements and using non-linearregression (Prism 5, Graphpad Software), the concentration of ephrin-A4in unknown samples was calculated. In this regard plasma samples of fourhealthy adults, four patients diagnosed with B cell chronic lymphocyticleukemia (B-CLL) and four patients diagnosed with multiple myeloma (MM)were analyzed for their secreted ephrin-A4 concentration. The dataobtained suggest that the hEFNA4 analyte be significantly higher in CLLpatients than in healthy adults or other selected B-cell derived tumors(FIG. 17B). Moreover, as previously indicted and shown in FIG. 17C,secreted hENFA4 is also detectable in mice harboring human colorectalcancer xenotransplants. Specifically in FIG. 17C each point isrepresentative of secreted hEFNA4 levels in sera obtained from adifferent mouse. Conversely, serum levels of secreted hEFNA4 innon-xenotransplanted mice were essentially undetectable (data notshown). Even more surprisingly, when plotting tumor volume against theconcentration of hEFNA4 in the serum samples, a significant correlationwas observed suggesting that the secreted analyte might be particularlyuseful to monitor tumor growth of certain human solid tumors in vivo.More generally, these results are strongly indicative as to theapplicability of the instant invention in both therapeutic anddiagnostic settings.

Using the above described method, plasma samples from 23 normal humandonors obtained from a blood bank were used to determine theconcentration range of this analyte in healthy adults. As shown in FIG.17D, a mean concentration of 332 pg/ml EFNA4 (6.2 pg/ml standarddeviation) was found. This indicates that EFNA is secreted or shed atvery low and tightly regulated concentration and makes EFNA an idealbiomarker or diagnostic marker to monitor disease progression ordiagnose EFNA associated disorders.

To explore this possibility further, commercially obtained serum samplesfrom 17 patients with colorectal cancer and 10 samples from patientswith non-small cell lung cancer were compared to 12 samples from healthyadults were tested for the EFNA4 concentration using the above describedmethod. As shown in FIG. 17E, both patients with colorectal cancer andnon-small cell lung cancer had significantly elevated circulating EFNA4levels in their blood. Using an unpaired t test, the comparison betweenhealthy adults and colorectal cancer patients reached a p-value of0.0002 and between healthy adults and non-small cell lung cancerpatients of 0.01. The data demonstrate that secreted or shed EFNA4 iselevated in patients with solid tumors and illustrates the value ofusing the disclosed modulators in analytical testing or clinicaldiagnostics.

Example 18 EFNA4 Modulators can Target Cells that Express Related EFNALigands

Ligand specificity of EFNA4 modulators was tested against related EFNAligands to evaluate the degree of cross-reactivity. As an example,SC4.2.1 and SC9.65 were tested in the in vitro killing assay usingHEK293T cells overexpressing EFNA4 (FIG. 17A), EFNA3 (FIG. 17B) andEFNA1 (FIG. 17C). Note that the modulator SC9.65 was generated byimmunizing mice with EFNA1 immunogen (as per Example 6). The killingassay was carried out just as described in Example 16. FIG. 17demonstrates that SC4.2.1 is able to kill EFNA3 in addition to EFNA4expressing cells, and SC9.65 is able to kill EFNA1 and EFNA4 expressingcells. These data illustrate that selected modulators generated againsta specific EFNA family member can bind other family members sufficientlywell to bind, induce internalization and deliver a cytotoxic payload toligand expressing cells. This discovery is somewhat unexpected given thelow degree of homology between EFNA family members (approximately 34-45%amino acid sequence identity between human EFNA1, 2, 3 and 4) andexemplifies that, as described herein, pan-EFNA modulators can begenerated for diagnostic or therapeutic purposes.

Example 19 EFNA Ligands Interact Selectively with Multiple EphAReceptors

As discussed above ephrin-A ligands are known to bind to numerous EphAreceptors. To explore which EphA receptors have the potential tointeract with EFNA4, a flow cytometric binding assay similar to the onedescribed in Example 9 was developed. More particularly soluble EphAreceptors expressed as human IgG1 Fc fusion constructs were added tofifty thousand HEK293T cells per well (FIG. 19A) or HEK293T cellsoverexpressing EFNA4 (FIG. 19B) by means of retroviral transduction(referred to as HEK293T.hEFNA4 cells) for 1 hour in staining buffer at4° C. After washing, a secondary anti-human IgG polyclonal antibodyconjugated to Dylight 649 (Jackson Immuno Research) was added for onehour. After washing twice, samples were resuspended in staining buffercontaining 2 μg/ml DAPI and analyzed on a FACS Canto II (BD Biosciences)under standard conditions using the HTS attachment. FIGS. 19A and 19Bdemonstrate that EphA2, EphA3, EphA4, EphA6, EphA7 and EphA10 but notEphA1 bind to ephrin-A4 ligand. This again points to the advantages andpotential multifaceted points of action inherent in modulators of theinstant invention.

Example 20 EFNA4 Binds to EphB2 but not EphB3 and EphB4 Receptors

Extending the finding shown in Example 20, the ability of ephrin-A4ligand to bind to EphB receptors was explored. EFNA4 was initiallyidentified as CSC associated target as demonstrated above in Examples2-4. In the tissue hierarchy of the normal mouse colon crypts, EphB2 andEphB3 receptors are highly expressed by cells residing at colon cryptbase and not by cells located at top of the crypt indicating that EphBexpression and forward or reverse signaling through EphB receptors isimportant in tissue organization and individual cell fate decisions(Batlle et al.; 2002 PMID:12408869). More recently, EphB2 expression bycolorectal cancer cells has been linked to tumor initiation andlong-term proliferative capabilities suggesting that EphB2 may serve asphenotypic marker for cancer stem cells of the colon (Merlos-Suarez etal., 2011 PMID: 21419747). Hence the ability of ephrin-A4 ligand to bindto any of the differentially expressed EphB receptors might be ofbiological importance of colorectal cancer stem cells.

Using art-recognized techniques soluble EphB receptors expressed ashuman IgG1 Fc fusion constructs as well as EphA1-Fc (which does not bindEFNA4) and EphA2-Fc (which does bind EFANA4 ligand strongly) were addedto fifty thousand HEK293T cells per well (FIG. 20A) or HEK293T.hEFNA4cells (FIG. 20B) by for 1 hour in staining buffer at 4° C. Afterwashing, a secondary anti-human IgG polyclonal antibody conjugated toDylight 649 (Jackson Immuno Research) was added for one hour. Afterwashing twice, samples were resuspended in staining buffer containing 2μg/ml DAPI and analyzed on a FACS Canto II (BD Biosciences) understandard conditions using the HTS attachment. FIGS. 20A and 20Bdemonstrate that EphB2 but not EphB3 and EphB4 bind to EFNA4 ligandagain emphasizing the potential diversity of therapeutic pathways thatmay be advantageously impacted by the disclosed modulators.

Those skilled in the art will further appreciate that the presentinvention may be embodied in other specific forms without departing fromthe spirit or central attributes thereof. In that the foregoingdescription of the present invention discloses only exemplaryembodiments thereof, it is to be understood that other variations arecontemplated as being within the scope of the present invention.Accordingly, the present invention is not limited to the particularembodiments that have been described in detail herein. Rather, referenceshould be made to the appended claims as indicative of the scope andcontent of the invention.

The invention claimed is:
 1. A method of treating an EFNA4-expressingneoplastic disorder comprising administering a therapeutically effectiveamount of a composition comprising an antibody or antigen-bindingfragment thereof that specifically binds to EFNA4 to a subject in needthereof, wherein the antibody or antigen-binding fragment thereofcomprises at least one heavy chain variable region and at least onelight chain variable region, wherein the at least one heavy chainvariable region comprises three CDRs of a heavy chain variable regionset forth as SEQ ID NO: 157 and wherein the at least one light chainvariable region comprises three CDRs of a light chain variable regionset forth as SEQ ID NO:
 159. 2. The method of claim 1, wherein theneoplastic disorder comprises a solid tumor.
 3. The method of claim 1,wherein the neoplastic disorder is breast cancer, ovarian cancer,colorectal cancer, liver cancer, or lung cancer.
 4. The method of claim3, wherein the neoplastic disorder is breast cancer.
 5. The method ofclaim 3, wherein the neoplastic disorder is ovarian cancer.
 6. Themethod of claim 1, wherein the neoplastic disorder is a hematologicmalignancy.
 7. The method of claim 6, wherein the hematologic malignancyis leukemia.
 8. The method of claim 1, wherein the antibody orantigen-binding fragment thereof comprises at least one heavy chainvariable region and at least one light chain variable region, whereinthe at least one heavy chain variable region comprises three CDRs setforth as residues 31-35 of SEQ ID NO: 157 for CDR-H1, residues 50-65 ofSEQ ID NO: 157 for CDR-H2, and residues 95-102 of SEQ ID NO: 157 forCDR-H3, and wherein the at least one light chain variable regioncomprises three CDRs set forth as residues 24-34 of SEQ ID NO: 159 forCDR-L1, residues 50-56 of SEQ ID NO: 159 for CDR-L2, and residues 89-97of SEQ ID NO: 159 for CDR-L3, wherein the residues are numberedaccording to Kabat.
 9. The method of claim 1, wherein the antibody orantigen-binding fragment thereof comprises at least one heavy chainvariable region and at least one light chain variable region, whereinthe at least one heavy chain variable region comprises three CDRs setforth as residues 26-32 of SEQ ID NO: 157 for CDR-H1, residues 53-55 ofSEQ ID NO: 157 for CDR-H2, and residues 96-101 of SEQ ID NO: 157 forCDR-H3, and wherein the at least one light chain variable regioncomprises three CDRs set forth as residues 26-32 of SEQ ID NO: 159 forCDR-L1, residues 50-52 of SEQ ID NO: 159 for CDR-L2, and residues 91-96of SEQ ID NO: 159 for CDR-L3, wherein the residues are numberedaccording to Chothia.
 10. The method of claim 1, wherein the antibody orantigen-binding fragment thereof comprises at least one heavy chainvariable region and at least one light chain variable region, whereinthe at least one heavy chain variable region comprises three CDRs setforth as residues 30-35 of SEQ ID NO: 157 for CDR-H1, residues 47-58 ofSEQ ID NO: 157 for CDR-H2, and residues 93-101 of SEQ ID NO: 157 forCDR-H3, and wherein the at least one light chain variable regioncomprises three CDRs set forth as residues 30-36 of SEQ ID NO: 159 forCDR-L1, residues 46-55 of SEQ ID NO: 159 for CDR-L2, and residues 89-96of SEQ ID NO: 159 for CDR-L3, wherein the residues are numberedaccording to MacCallum.
 11. The method of claim 1, wherein the antibodyor antigen-binding fragment thereof comprises a heavy chain variableregion set forth as SEQ ID NO: 157 and a light chain variable region setforth as SEQ ID NO:
 159. 12. The method of claim 1, wherein the antibodyor antigen-binding fragment thereof is conjugated, linked or otherwiseassociated with a cytotoxic agent.
 13. The method of claim 11, whereinthe antibody or antigen-binding fragment thereof is conjugated, linkedor otherwise associated with a cytotoxic agent.
 14. A method of treatingan EFNA4-expressing cancer comprising administering a therapeuticallyeffective amount of a composition comprising an antibody orantigen-binding fragment thereof that specifically binds to EFNA4 to asubject in need thereof, wherein the antibody or antigen-bindingfragment thereof comprises at least one heavy chain variable region andat least one light chain variable region, wherein the at least one heavychain variable region comprises three CDRs of a heavy chain variableregion set forth as SEQ ID NO: 157 and wherein the at least one lightchain variable region comprises three CDRs of a light chain variableregion set forth as SEQ ID NO:
 159. 15. The method of claim 14, whereinthe cancer comprises a solid tumor.
 16. The method of claim 14, whereinthe cancer is breast cancer, ovarian cancer, colorectal cancer, livercancer, or lung cancer.
 17. The method of claim 16, wherein the canceris breast cancer.
 18. The method of claim 16, wherein the cancer isovarian cancer.
 19. The method of claim 14, wherein the cancer is ahematologic malignancy.
 20. The method of claim 19, wherein thehematologic malignancy is leukemia.
 21. The method of claim 14, whereinthe antibody or antigen-binding fragment thereof comprises at least oneheavy chain variable region and at least one light chain variableregion, wherein the at least one heavy chain variable region comprisesthree CDRs set forth as residues 31-35 of SEQ ID NO: 157 for CDR-H1,residues 50-65 of SEQ ID NO: 157 for CDR-H2, and residues 95-102 of SEQID NO: 157 for CDR-H3, and wherein the at least one light chain variableregion comprises three CDRs set forth as residues 24-34 of SEQ ID NO:159 for CDR-L1, residues 50-56 of SEQ ID NO: 159 for CDR-L2, andresidues 89-97 of SEQ ID NO: 159 for CDR-L3, wherein the residues arenumbered according to Kabat.
 22. The method of claim 14, wherein theantibody or antigen-binding fragment thereof comprises at least oneheavy chain variable region and at least one light chain variableregion, wherein the at least one heavy chain variable region comprisesthree CDRs set forth as residues 26-32 of SEQ ID NO: 157 for CDR-H1,residues 53-55 of SEQ ID NO: 157 for CDR-H2, and residues 96-101 of SEQID NO: 157 for CDR-H3, and wherein the at least one light chain variableregion comprises three CDRs set forth as residues 26-32 of SEQ ID NO:159 for CDR-L1, residues 50-52 of SEQ ID NO: 159 for CDR-L2, andresidues 91-96 of SEQ ID NO: 159 for CDR-L3, wherein the residues arenumbered according to Chothia.
 23. The method of claim 14, wherein theantibody or antigen-binding fragment thereof comprises at least oneheavy chain variable region and at least one light chain variableregion, wherein the at least one heavy chain variable region comprisesthree CDRs set forth as residues 30-35 of SEQ ID NO: 157 for CDR-H1,residues 47-58 of SEQ ID NO: 157 for CDR-H2, and residues 93-101 of SEQID NO: 157 for CDR-H3, and wherein the at least one light chain variableregion comprises three CDRs set forth as residues 30-36 of SEQ ID NO:159 for CDR-L1, residues 46-55 of SEQ ID NO: 159 for CDR-L2, andresidues 89-96 of SEQ ID NO: 159 for CDR-L3, wherein the residues arenumbered according to MacCallum.
 24. The method of claim 14, wherein theantibody or antigen-binding fragment thereof comprises a heavy chainvariable region set forth as SEQ ID NO: 157 and a light chain variableregion set forth as SEQ ID NO:
 159. 25. The method of claim 14, whereinthe antibody or antigen-binding fragment thereof is conjugated, linkedor otherwise associated with a cytotoxic agent.
 26. The method of claim24, wherein the antibody or antigen-binding fragment thereof isconjugated, linked or otherwise associated with a cytotoxic agent.
 27. Amethod of treating an EFNA4-expressing neoplastic disorder comprisingadministering a therapeutically effective amount of a compositioncomprising an antibody or antigen-binding fragment thereof thatspecifically binds to EFNA4 to a subject in need thereof, wherein theantibody or antigen-binding fragment thereof comprises three CDRs of aheavy chain variable region set forth as SEQ ID NO: 157 and three CDRsof a light chain variable region set forth as SEQ ID NO: 159, whereinsaid EFNA4-expressing neoplastic disorder is breast cancer, ovariancancer, colorectal cancer, liver cancer, or lung cancer.